Inactivation Of Rb Research Articles

The G1-S transition is promoted by Rb degradation via the E3 ligase UBR5.

Mammalian cells make the decision to divide at the G1-S transition in response to diverse signals impinging on the retinoblastoma protein Rb, a cell cycle inhibitor and tumor suppressor. Passage through the G1-S transition is initially driven by Rb inactivation via phosphorylation and by Rb's decreasing concentration in G1. While many studies have identified the mechanisms of Rb phosphorylation, the mechanism underlying Rb's decreasing concentration in G1 was unknown. Here, we found that Rb's concentration decrease in G1 requires the E3 ubiquitin ligase UBR5. UBR5 knockout cells have increased Rb concentration in early G1, exhibited a lower G1-S transition rate, and are more sensitive to inhibition of cyclin-dependent kinase 4/6 (Cdk4/6). This last observation suggests that UBR5 inhibition can strengthen the efficacy of Cdk4/6 inhibitor-based cancer therapies.

Pre-ciliated tubal epithelial cells are prone to initiation of high-grade serous ovarian carcinoma

The distal region of the uterine (Fallopian) tube is commonly associated with high-grade serous carcinoma (HGSC), the predominant and most aggressive form of ovarian or extra-uterine cancer. Specific cell states and lineage dynamics of the adult tubal epithelium (TE) remain insufficiently understood, hindering efforts to determine the cell of origin for HGSC. Here, we report a comprehensive census of cell types and states of the mouse uterine tube. We show that distal TE cells expressing the stem/progenitor cell marker Slc1a3 can differentiate into both secretory (Ovgp1+) and ciliated (Fam183b+) cells. Inactivation of Trp53 and Rb1, whose pathways are commonly altered in HGSC, leads to elimination of targeted Slc1a3+ cells by apoptosis, thereby preventing their malignant transformation. In contrast, pre-ciliated cells (Krt5+, Prom1+, Trp73+) remain cancer-prone and give rise to serous tubal intraepithelial carcinomas and overt HGSC. These findings identify transitional pre-ciliated cells as a cancer-prone cell state and point to pre-ciliation mechanisms as diagnostic and therapeutic targets.

P16 Immunohistochemical Patterns in Triple-Negative Breast Cancer: Clinical and Genomic Similarities of the p16 Diffuse Pattern to pRB Deficiency

Introduction: Triple-negative breast cancer (TNBC) is associated with alterations in the retinoblastoma pathway. As a consequence of retinoblastoma protein (pRB) loss, compensatory upregulation of p16 occurs due to the loss of phosphorylated pRB-mediated negative feedback on p16 expression. The aim of this study is to investigate the clinicopathological and genomic characteristics associated with the diffuse pattern of p16 immunohistochemistry (IHC) in TNBC. Methods: The study analyzed surgically resected TNBC for whole-exome sequencing in 113 cases and for cDNA microarray in 144 cases. The p16 IHC results were classified into two patterns: diffuse and negative/mosaic. Results: In the entire cohort (n = 257), the diffuse pattern of p16 IHC was observed in 123 (47.9%) patients and the negative/mosaic pattern in 134 (52.1%). Biallelic RB1 inactivation was observed in 14.3% of patients with the diffuse pattern. The diffuse pattern of p16 IHC showed more frequent RB1 alterations and cell cycle progression signatures, a higher Ki-67 labeling index, more frequent chromosome segment copy number changes, a higher frequency of homologous recombination deficiency high, and immune-related signatures. PIK3CA mutations were more frequent in the negative/mosaic pattern. CCND1 amplification was identified in 5 cases, all with the negative/mosaic pattern. Conclusion: In TNBC, the diffuse p16 pattern shows clinical and genomic similarities to pRB-deficient tumors, suggesting shared characteristics. This suggests that p16 IHC testing may provide new therapeutic approaches, underscoring its potential clinical importance.

Chromothripsis-mediated small cell lung carcinoma.

Small cell lung carcinoma (SCLC) is a highly aggressive malignancy that is typically associated with tobacco exposure and inactivation of RB1 and TP53 genes. Here we performed detailed clinicopathologic, genomic and transcriptomic profiling of an atypical subset of SCLC that lacked RB1 and TP53 co-inactivation and arose in never/light smokers. We found that most cases were associated with chromothripsis - massive, localized chromosome shattering - recurrently involving chromosomes 11 or 12, and resulting in extrachromosomal (ecDNA) amplification of CCND1 or co-amplification of CCND2/CDK4/MDM2, respectively. Uniquely, these clinically aggressive tumors exhibited genomic and pathologic links to pulmonary carcinoids, suggesting a previously uncharacterized mode of SCLC pathogenesis via transformation from lower-grade neuroendocrine tumors or their progenitors. Conversely, SCLC in never-smokers harboring inactivated RB1 and TP53 exhibited hallmarks of adenocarcinoma-to-SCLC derivation, supporting two distinct pathways of plasticity-mediated pathogenesis of SCLC in never-smokers.

Comprehensive identification of pathogenic variants in retinoblastoma by long- and short-read sequencing

Retinoblastoma (RB) is the most common intraocular malignancy in childhood. The causal variants in RB are mostly characterized by previously used short-read sequencing (SRS) analysis, which has technical limitations in identifying structural variants (SVs) and phasing information. Long-read sequencing (LRS) technology has advantages over SRS in detecting SVs, phased genetic variants, and methylation. In this study, we comprehensively characterized the genetic landscape of RB using combinatorial LRS and SRS of 16 RB tumors and 16 matched blood samples. We detected a total of 232 somatic SVs, with an average of 14.5 SVs per sample across the whole genome in our cohort. We identified 20 distinct pathogenic variants disrupting RB1 gene, including three novel small variants and five somatic SVs. We found more somatic SVs were detected from LRS than SRS (140 vs. 122) in RB samples with WGS data, particularly the insertions (18 vs. 1). Furthermore, our analysis shows that, with the exception of one sample who lacked the methylation data, all samples presented biallelic inactivation of RB1 in various forms, including two cases with the biallelic hypermethylated promoter and four cases with compound heterozygous mutations which were missing in SRS analysis. By inferring relative timing of somatic events, we reveal the genetic progression that RB1 disruption early and followed by copy number changes, including amplifications of Chr2p and deletions of Chr16q, during RB tumorigenesis. Altogether, we characterize the comprehensive genetic landscape of RB, providing novel insights into the genetic alterations and mechanisms contributing to RB initiation and development. Our work also establishes a framework to analyze genomic landscape of cancers based on LRS data.

Targeting Tyro3, Axl, and MerTK Receptor Tyrosine Kinases Significantly Sensitizes Triple-Negative Breast Cancer to CDK4/6 Inhibition.

Triple-negative breast cancer (TNBC) is the most aggressive subtype with high metastasis and mortality rates. Given the lack of actionable targets such as ER and HER2, TNBC still remains an unmet therapeutic challenge. Despite harboring high CDK4/6 expression levels, the efficacy of CDK4/6 inhibition in TNBC has been limited due to the emergence of resistance. The resistance to CDK4/6 inhibition is mainly mediated by RB1 inactivation. Since our aim is to overcome resistance to CDK4/6 inhibition, in this study, we primarily used the cell lines that do not express RB1. Following a screening for activated receptor tyrosine kinases (RTKs) upon CDK4/6 inhibition, we identified the TAM (Tyro3, Axl, and MerTK) RTKs as a crucial therapeutic vulnerability in TNBC. We show that targeting the TAM receptors with a novel inhibitor, sitravatinib, significantly sensitizes TNBC to CDK4/6 inhibitors. Upon prolonged HER2 inhibitor treatment, HER2+ breast cancers suppress HER2 expression, physiologically transforming into TNBC-like cells. We further show that the combined treatment is highly effective against drug-resistant HER2+ breast cancer as well. Following quantitative proteomics and RNA-seq data analysis, we extended our study into the immunophenotyping of TNBC. Given the roles of the TAM receptors in promoting the creation of an immunosuppressive tumor microenvironment (TME), we further demonstrate that the combination of CDK4/6 inhibitor abemaciclib and sitravatinib modifies the immune landscape of TNBC to favor immune checkpoint blockade. Overall, our study offers a novel and highly effective combination therapy against TNBC and potentially treatment-resistant HER2+ breast cancer that can be rapidly moved to the clinic.

RB1 Genetic Alterations in Estrogen Receptor–Positive Breast Carcinomas: Correlation With Neuroendocrine Differentiation

Genetic alterations in the retinoblastoma susceptibility gene (RB1) are present in up to 40% of triple-negative breast cancers (BCs) and frequent in tumors with neuroendocrine differentiation, including small cell neuroendocrine carcinoma. Data on RB1 genetic alterations in estrogen receptor (ER)–positive BCs are scarce. In this study, we sought to define the morphologic, immunohistochemical, and genetic features of ER-positive BCs harboring somatic alterations in RB1, with emphasis on neuroendocrine differentiation. ER-positive BCs with pathogenic RB1 genetic alterations were identified in <1% of cases (N = 55) from a cohort of 6026 BCs previously subjected to targeted next-generation sequencing, including 23 primary BCs (pBCs) and 32 recurrent/metastatic BCs (mBCs). In cases where loss of heterozygosity of the wild-type RB1 allele could be assessed (93%, 51/55), most pBCs (82%, 18/22) and mBCs (90%, 26/29) exhibited biallelic RB1 inactivation, primarily through loss-of-function mutation and loss of heterozygosity (98%, 43/44). Upon histologic review, a subset of RB1-altered tumors exhibited neuroendocrine morphology (13%, 7/55), which correlated with expression of neuroendocrine markers (39%, 9/23) in both pBCs (27%, 3/11) and mBCs (50%, 6/12). Loss of Rb protein expression was observed in BCs with biallelic RB1 loss only, with similar frequency in pBCs (82%, 9/11) and mBCs (75%, 9/12). All cases with neuroendocrine marker expression (n = 9) and/or neuroendocrine morphology (n = 7) harbored biallelic genetic inactivation of RB1 and exhibited Rb loss of expression. TP53 (53%, 29/55) and PIK3CA (45%, 25/55) were the most frequently comutated genes across the cohort. Overall, these findings suggest that ER-positive BCs with biallelic RB1 genetic alterations frequently exhibit Rb protein loss, which correlates with neuroendocrine differentiation in select BCs. This study provides insights into the molecular and phenotypic heterogeneity of BCs with RB1 genetic inactivation, underscoring the need for further research into the potential clinical implications associated with these tumors.

Abstract PO3-23-10: Continued treatment with CDK4/6 inhibitors slow tumor progression by extending G1-phase duration in drug resistant populations

Abstract CDK4/6 inhibitors (CDK4/6i) have significantly improved the prognosis of patients with metastatic hormone receptor-positive breast cancer. However, the development of drug resistance leads to the discontinuation of CDK4/6i treatment. The potential benefits of maintaining CDK4/6i treatment in the context of resistance and their underlying molecular mechanisms remain elusive. Here we show that despite tumor progression, continued treatment with CDK4/6i notably extends the G1 phase in drug-resistant cells, thereby slowing their growth. Our single-cell data reveal that the duration of the G1 phase in these resistant cells is approximately tripled under continuous CDK4/6i treatment compared to conditions with CDK4/6i withdrawal, while the durations of the S and G2 phases remain comparable. The maintenance of CDK4/6i induces an alternative inactivation of the retinoblastoma (Rb) protein in drug-resistant cells through its post-translational degradation. However, this alternative form of Rb inactivation is ineffective and thus induces slow activation kinetics of E2F transcription factors, significantly delaying G1 progression. The beneficial effect of maintaining CDK4/6i treatment was further validated in breast cancer xenograft models. Furthermore, our findings suggest that given the spectrum of various CDK4/6i, switching between different CDK4/6i types after the emergence of drug resistance may further delay tumor progression. This study provides new mechanistic insights into the maintenance of CDK4/6i treatment despite the development of drug resistance. Citation Format: Jessica Armand, Sungsoo Kim, Hee Won Yang. Continued treatment with CDK4/6 inhibitors slow tumor progression by extending G1-phase duration in drug resistant populations [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-23-10.

Abstract 2004: CDC7 inhibition prevents neuroendocrine transformation in the lung and prostate through MYC degradation

Abstract Neuroendocrine (NE) transformation occurs as a mechanism of resistance to targeted therapy in up to 14% and 30% of EGFR-mutant lung and AR-dependent prostate adenocarcinomas, respectively, leading to poor prognosis. Even if we know the tumor population at high risk of transformation (TP53/RB1-mutated), no therapies to prevent NE relapse are currently available. To identify therapeutic vulnerabilities for tumors undergoing NE transformation, we performed an in vitro CRISPR screen in a NE-transformed lung tumor model. This screen identified CDC7, involved in DNA replication and DNA damage response, as a potential therapeutic target in this setting. Proteogenomic analyses revealed CDC7 upregulation in lung and prostate clinical samples undergoing NE transformation, detected already in pre-transformation adenocarcinomas. These results indicated that CDC7 expression is induced already at early steps of transformation. Consistently, TP53/RB1-mutated lung and prostate adenocarcinomas exhibited higher CDC7 expression than their double wild-type counterparts. Combined ChIP-seq and promoter reporter assays indicated that CDC7 expression was directly regulated by TP53 and RB1 inactivation. Importantly, TP53/RB1-inactivation induced sensitivity to the CDC7 inhibitor simurosertib, unraveling a therapeutic vulnerability in tumors at high risk of NE transformation. Thus, we tested the combination of simurosertib with targeted therapy in vivo in different lung and prostate patient-derived models of NE transformation, namely TP53/RB1-knock out adenocarcinomas known to undergo NE transformation on targeted therapy. In these, simurosertib was able to suppress NE transformation and dramatically delay tumor relapse. Trajectory analysis on single-cell transcriptomic data for such models revealed a NE transformation transcriptional program occurring already in the untreated tumors before transformation. Remarkably, simurosertib treatment reverted this transcriptomic state and induced a reversion to the original adenocarcinoma transcriptomic profile. CDC7 inhibition led to increased proteasomal activity and degradation of MYC, a stemness transcription factor involved in NE transformation. Ectopic overexpression of MYCT58A, a proteasome degradation-resistant MYC isoform, rescued the NE phenotype in these transformation models, suggesting that CDC7 inhibition-induced MYC degradation is the mechanism by which NE transformation is prevented. In sum, CDC7 inhibition may suppress, or at least dramatically delay NE transformation in patients with lung and prostate adenocarcinomas at high risk of transformation, by inducing MYC proteasomal degradation. The clinical availability of CDC7 inhibitors, currently in phase II clinical trials after demonstrating tolerability and preliminary efficacy, will allow rapid translation of these results into the clinics. Citation Format: Alvaro Quintanal-Villalonga, Fathema Uddin, Kenta Kawasaki, Esther Redin, Vidushi Durani, Amin Sabet, Wouter Karthaus, Yingqian A. Zhan, Samir Zaidi, Moniquetta Shaffer, Harsha Sridhar, Juan Qiu, Parvathy Manoj, Elisa De Stanchina, Michael C. Haffner, Charles L. Sawyers, Charles M. Rudin. CDC7 inhibition prevents neuroendocrine transformation in the lung and prostate through MYC degradation [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 2004.

Abstract 2551: Harness the power of real-world-data for oncology target and biomarker research: A case study by applying clinic-genomics data for 2,659 lung cancer patients and beyond

Abstract Randomized controlled trials (RCTs) are the gold standard for evaluating drug safety and efficacy in controlled settings, but they have limited generalizability due to their focus on selective and homogeneous populations. Real-world-data (RWD), collected from larger and more varied patient populations, can address inherent limitations of RCTs and aid in clinical trial design and drug approval. However, with the challenges of RWD, unlocking its full potential for tumor target and biomarker identification and validation demands innovative analytical approaches. Here we present RWD analyses of ConcertAI® electronic health records (EHR) linked with Caris Lab data for 2,659 lung cancer patients. Comprehensive genomics analyses incorporate WES and RNA-seq data, coupled with rich clinical EHR data. By integrating clinical and genomics data, we showcase the potential of harnessing RWD as follows: (1) we investigated genomic alterations across five histology groups (adenocarcinoma, squamous, large cell, small cell lung cancer [SCLC], neuroendocrine [NE] lung cancer). Notably, TP53 and LRP1B mutations were prevalent across all groups. LRP1B mutations correlated with higher tumor mutation burden, indicative of a favorable response to immunotherapy. Biallelic TP53 and RB1 inactivation was predominant in SCLC and NE lung tumors. The five histological groups exhibit distinct expression profiles, reflecting tumor intrinsic characteristics. Most immune gene signature scores were significantly lower in SCLC and NE tumors, aligned with that they are cold tumors with immune resistance, except CD56-centered NK and neuroendocrine gene signatures; (2) we analyzed patient treatment patterns and persistence by line of therapy. Among the 206 NSCLC patients with clinically actionable EGFR mutations, 143 received targeted therapy during their treatment courses (69.4%). Time to treatment discontinuation (TTD) was used as a surrogate clinical outcome endpoint. TTD was significantly longer when TKI was the first line treatment compared to later lines (p = 0.01), while the trend is opposite for ICI where TTD is significantly shorter in the 1st line setting (p = 0.025); (3) we highlight the unique potential of leveraging H&amp;E imaging modality in such RWD. In summary, we successfully identified unique molecular signatures among various lung cancer histological groups, pinpointed distinct subpopulations within lung adenocarcinoma and squamous tumors linked to their immune phenotypes, and investigated the different patient treatment patterns and outcomes, facilitating personalized medicine. In the broader context of our work, we have developed a framework for harnessing various data modalities (clinical, multi-omics, imaging) within AbbVie's oncology RWD realm, enhancing the discovery and validation of new oncology targets and biomarkers. Citation Format: Si Wu, Weilong Zhao, Christy Choi, Mona Cai, Peter Ansell, Alexander Liede, Rong Chen, Xi Zhao, Josue Samayoa. Harness the power of real-world-data for oncology target and biomarker research: A case study by applying clinic-genomics data for 2,659 lung cancer patients and beyond [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 2551.

Abstract 3347: KIFC1 inhibition elicits antineoplastic activity in small cell lung cancer by inducing anaphase catastrophe

Abstract Cancer cells often have more than two centrosomes (supernumerary centrosomes) and this is one of the hallmarks of cancer. We previously demonstrated that CDK2 antagonism inhibits clustering of supernumerary centrosomes at mitosis leading to multipolar cell division and apoptotic death in non-small cell lung cancer. This antineoplastic process was designated anaphase catastrophe. Anaphase catastrophe can preferentially affect cancer cells sparing normal cells with two centrosomes. On the other hand, as CDK2 functions in normal cell cycle progression, its inhibition can also affect normal and malignant cells. Identification of a novel drug target that would preferentially cause anaphase catastrophe is needed. The motor protein KIFC1 affects centrosome clustering. Small cell lung cancer (SCLC), known for its poor prognosis with limited therapeutic options, displays disruptions in the cell cycle checkpoint mechanism due to inactivation of p53 and RB. We hypothesized SCLC was susceptible to mitotic abnormalities, including anaphase catastrophe. In this study, we explored the possibility of KIFC1 as a novel therapeutic target that triggers anaphase catastrophe in SCLC. In-silico GEO database analysis revealed elevated levels of KIFC1 and PLK4 in SCLCs compared with other cancers and normal tissues. PLK4 plays a key role in centrosome amplification. Our in-silico analysis results imply that centrosome amplification and their clustering frequently occur in SCLC. We performed in-vitro functional analysis of KIFC1 inhibition in SCLC cell lines (NCI-H146, SHP77, NCI-H209, and NCI-H524 cells), using siRNAs and CRISPR-Cas9 system for genetic inhibition and AZ82, a specific KIFC1 inhibitor, for pharmacologic inhibition of KIFC1. Cell proliferation was measured by the CellTiter-Glo Luminescent Assay and apoptosis was scored by annexin V and PI staining followed by flow cytometry analysis. Cell growth was reduced and apoptosis was statistically significantly increased after genetic inhibition of KIFC1 by siRNAs and by use of the CRISPR-Cas9 system in SCLC cell lines. These antineoplastic effects also occurred after AZ82 treatments, in a dose-dependent manner in these SCLC cell lines. To examine the mitotic status of SCLC cells after KIFC1 inhibition, SCLC cells were stained with α-tubulin antibody and DAPI before analysis with a fluorescence microscope. Besides normal bipolar mitotic cells, multipolar mitotic cells were observed, and their population was increased after KIFC1 knockdown with siRNAs. This indicated that clustering of supernumerary centrosomes was inhibited by targeting KIFC1 in SCLC cells. Taken together, KIFC1 is a potential therapeutic target to induce anaphase catastrophe in cancer cells. Targeting KIFC1 is a novel therapeutic strategy to consider for SCLC that currently has limited treatment options. Citation Format: Natsuki Nakagawa, Masakatsu Tokunaga, Mirei Ka, Yuriko Sugiura, Takahiro Iida, Takahiro Ando, Kousuke Watanabe, Xi Liu, Ethan Dmitrovsky, Hidenori Kage, Masanori Kawakami. KIFC1 inhibition elicits antineoplastic activity in small cell lung cancer by inducing anaphase catastrophe [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 3347.

Abstract 1959: BLU-222, a potent and highly selective CDK2 inhibitor, demonstrates antitumor activity as monotherapy and as combination treatment in CCNE1-aberrant endometrial cancer models

Abstract Background: Many cyclin-dependent kinases (CDK) bind regulatory cyclins to control cell cycle progression. Aberrant CDK2 activation occurs in selected cancers (e.g., ovarian cancer, endometrial cancer) through CCNE1 amplification, leading to Rb phosphorylation, inactivation, and cell cycle checkpoint dysregulation. CCNE1 amplification and subsequent cyclin E overexpression are associated with chemotherapy resistance and shorter progression-free and overall survival. Therefore, novel therapies are needed to inhibit aberrant CDK2 activation and elicit positive clinical benefit in these patients. BLU-222 is a potent, highly selective, orally bioavailable, investigational CDK2 inhibitor with demonstrated activity in preclinical CCNE1-amplified ovarian cancer models. Here, we explored additional, specific biomarkers to predict BLU-222 sensitivity in ovarian and endometrial cancer as monotherapy or in novel combination treatment strategies. Methods: In vitro antiproliferative effect of BLU-222 was assessed by CyQuant in a panel of CCNE1-aberrant or CCNE1-normal ovarian and endometrial cell lines. Combination of BLU-222 and paclitaxel was evaluated by dosing matrix; synergy was calculated using SynergyFinder. In vivo antitumor activity of BLU-222 as a single agent or combined with paclitaxel was measured in CCNE1-aberrant endometrial cancer patient-derived xenograft (PDX) models (XenoSTART). Results: In ovarian and endometrial cell lines, CCNE1 amplification (copy number ≥ 6) predicted strong antiproliferative response to BLU-222. Using multivariate biomarker analysis, high p16 protein expression and wild-type Rb protein were identified as additional predictive markers for response to BLU-222 in CCNE1-aberrant cell lines. BLU-222 with paclitaxel was broadly active in endometrial cancer cell lines, with the strongest combination effect in cells with high CCNE1 expression, wild-type Rb protein, and low p16 protein expression. In vivo, BLU-222 elicited strong monotherapy antitumor response in CCNE1 copy number-aberrant endometrial cancer PDX models, and this effect was strengthened with the combination of paclitaxel. Conclusions: In preclinical models of CCNE1-aberrant endometrial cancer, BLU-222 demonstrated enhanced activity in combination with paclitaxel. Furthermore, Rb and p16 protein expression can be used in conjunction with CCNE1 copy number to predict response to both BLU-222 monotherapy and combination treatment. These data provide a rationale for using BLU-222 in combination with paclitaxel and demonstrate the potential benefit of using specific biomarkers to predict response. Citation Format: Nealia House, Victoria Brown, Liang Yuan, Maxine Chen, Stephanie Lee, Rentian Wu, Lakshmi Muthuswamy, Scott Ribich, Philip Ramsden, Kerrie Faia. BLU-222, a potent and highly selective CDK2 inhibitor, demonstrates antitumor activity as monotherapy and as combination treatment in CCNE1-aberrant endometrial cancer models [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 1959.

Abstract 1632: Dissecting AMBRA1-cyclin D axis deregulation in embryonal tumors of the central nervous system

Abstract Dissecting AMBRA1-cyclin D axis deregulation in embryonal tumors of the centralnervous systemEmbryonal tumors of the central nervous system (CNS) are malignant growths originating from embryonic neural stem cells, primarily affecting pediatric patients. These tumors pose a significant threat to children's health, leading to high morbidity and mortality rates. To improve survival rates and reduce morbidity, there is a critical need for advanced molecular diagnostics and innovative therapies. Among these tumors are medulloblastomas (MB) and other embryonal tumors, previously classified as primitive neuroectodermal tumors (CNS-PNET). These malignancies are characterized by uncontrolled cell proliferation, primarily driven by abnormalities in cell cycle proteins. Consequently, targeting cell cycle regulators has emerged as a promising approach for their treatment. Two key signaling pathways, namely the MYC and the cyclin D-CDK-RB pathway, govern the decision-making process related to genome replication. Mutations in both MYC and the CDK4/6/cyclin D pathway, leading to the functional inactivation of RB, have been identified as relevant factors in the development of CNS-PNET and medulloblastomas. Our seminal research has highlighted the crucial role of AMBRA1, an upstream master regulator orchestrating the transition from G1 to S phase (Maiani &amp; Milletti et al., 2021). Specifically, AMBRA1 modulates the abundance of D-type cyclins and MYC by facilitating their proteasomal degradation, thereby ensuring genomic stability during DNA replication.Based on these discoveries, we propose that AMBRA1 functions as a multifaceted tumor suppressor across various cancer types. An unbiased Sleeping Beauty analysis identified AMBRA1 as a genetic driver of CNS-PNET, providing a strong foundation for investigating its role further (Beckmann et al., 2019). We hypothesize that the simultaneous deletion of AMBRA1 and p53 in an inducible knockout mouse model can lead to the spontaneous development of CNS-PNET or medulloblastoma, contingent on the specific targeted cell lineage. Additionally, our research demonstrates that AMBRA1 deficiency confers resistance to CDK4/6 inhibitors while creating a synthetic lethality with CHK1 inhibitors. Consequently, we propose that a comparative analysis of AMBRA1, D-type cyclins, and Myc family expression levels could serve as a diagnostic marker. This marker could facilitate patient-based stratification for CHK1 inhibitor treatment efficacy, enabling the differentiation of non- responsive cases to CDK4/6 chemotherapy. Citation Format: Giacomo Milletti, Giulia Cadeddu, Alba Adelantado Rubio, Caterina Ferraina, Apolinar Maya-Mendoza, Francesco Cecconi. Dissecting AMBRA1-cyclin D axis deregulation in embryonal tumors of the central nervous system [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 1632.

Abstract 236: Modeling multi-step retinoblastoma genesis with cone reporter retinal organoids

Abstract Purpose: This study aims to develop a human retinoblastoma organoid (RBRO) model that recapitulates the cell-of-origin and multi-step retinoblastoma genesis. Background: Retinoblastomas originate from maturing cone photoreceptor precursors with biallelic RB1 inactivation. Rb1 mutant animal models fail to recapitulate retinoblastomagenesis with a cone precursor cell-of-origin, likely due to human-specific cone development features. In explanted fetal retina, pRB-depleted post-mitotic cone precursors proliferate, followed by a 3-5 month premalignant indolence phase before retinoblastoma-like masses emerge at tissue ages similar to retinoblastomas in vivo. However, tissue availability limits research with this disease model. RB1−/- retinal organoids (ROs) provide a potential alternative, as they demonstrate cone proliferation, but they deteriorate before forming indolent premalignant lesions or malignant retinoblastoma foci. Here, we developed methods with which to examine the effects of cone precursor pRB loss in otherwise healthy retinal tissue. Methods: We generated cone-reporter GNAT2-EGFP iPSC lines through CRISPR knock-in of EGFP-P2A at the N-terminus of GNAT2 in WTC11-mTagRFPT-LMNB1. A second round of CRISPR editing produced homozygous RB1 knockout. Chimeric RB1 WT ROs and RB1-null RBROs were generated from cone reporter iPSCs mixed with unedited parental iPSCs. ROs and RBROs were embedded in hydrogel and live-imaged episodically to track EGFP+ cone proliferation dynamics. scRNA-seq was carried out on FACS isolated EGFP+ RB1-null cones at various ages. Results: In RB1 WT ROs, EGFP+ cone precursors appeared at d34 and adopted cone morphology at ~d120, with rapid inner segment growth between ~d120 and ~d150. Immunohistochemistry confirmed cone-specific EGFP expression. In RB1-null RBROs, bi-weekly live confocal imaging revealed initial EGFP+ RB1−/− cone proliferation followed by a pre-malignant indolence phase starting at ~d150. The majority of the initially proliferating cones were Ki67-negative with some adopting mature cone photoreceptor morphology. Nascent retinoblastoma-like foci co-expressing EGFP, cone markers, and Ki67 formed in five of six RBROs after d281, a tissue age that equates to the first post-natal month when early retinoblastomas typically emerge. scRNA-seq of EGFP+ RB1−/− cones from the initial proliferation, indolence, and retinoblastoma-like stages revealed distinct molecular signatures. Conclusions: We generated a human retinoblastoma organoid model that recapitulates the cell-of-origin and timing of multi-step retinoblastomagenesis, with each tumorigenesis stage harboring distinct molecular signatures. This model may enable the identification of epigenetic and transcriptomic changes underlying malignant progression and screening for compounds that efficiently block this process. Citation Format: Jinlun Bai, David S. Koos, Kayla Stepanian, Kevin Stachelek, Bhavana Bhat, Scott Fraser, Rex Moats, David Cobrinik. Modeling multi-step retinoblastoma genesis with cone reporter retinal organoids [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 236.

Abstract PR-010: Asc1lb progenitor-specific RB conditional inactivation in zebrafish models rare CNS primitive neuroectodermal tumors

Abstract PR-010: Asc1lb progenitor-specific RB conditional inactivation in zebrafish models rare CNS primitive neuroectodermal tumors

Genomic and transcriptomic profiling of combined small-cell lung cancer through microdissection: unveiling the transformational pathway of mixed subtype.

Combined small-cell lung carcinoma (cSCLC) represents a rare subtype of SCLC, the mechanisms governing the evolution of cancer genomes and their impact on the tumor immune microenvironment (TIME) within distinct components of cSCLC remain elusive. Here, we conducted whole-exome and RNA sequencing on 32 samples from 16 cSCLC cases. We found striking similarities between two components of cSCLC-LCC/LCNEC (SCLC combined with large-cell carcinoma/neuroendocrine) in terms of tumor mutation burden (TMB), tumor neoantigen burden (TNB), clonality structure, chromosomal instability (CIN), and low levels of immune cell infiltration. In contrast, the two components of cSCLC-ADC/SCC (SCLC combined with adenocarcinoma/squamous-cell carcinoma) exhibited a high level of tumor heterogeneity. Our investigation revealed that cSCLC originated from a monoclonal source, with two potential transformation modes: from SCLC to SCC (mode 1) and from ADC to SCLC (mode 2). Therefore, cSCLC might represent an intermediate state, potentially evolving into another histological tumor morphology through interactions between tumor and TIME surrounding it. Intriguingly, RB1 inactivation emerged as a factor influencing TIME heterogeneity in cSCLC, possibly through neoantigen depletion. Together, these findings delved into the clonal origin and TIME heterogeneity of different components in cSCLC, shedding new light on the evolutionary processes underlying this enigmatic subtype.

Abstract PR04: USP7 inhibitors prevent triple negative breast cancer metastasis by inducing UHRF1 protein degradation

Abstract Functional inactivation of RB1 occurs frequently in triple negative breast cancer (TNBC), the most aggressive breast cancer subtype with poor clinical prognosis when tumors metastasize. Ubiquitin-like with PHD and RING Finger domains 1 (UHRF1) is a key epigenetic regulator that is controlled by the RB-E2F pathway and is frequently overexpressed in many types of cancers, including TNBC. Using TCGA and METABRIC databases, we found that UHRF1 overexpression is negatively associated with breast cancer patient survival, including TNBC. Genetic ablation of UHRF1 using CRISPR/Cas9 in TNBC cell lines resulted in a robust decrease in tumorigenicity, including decreased cell clonogenicity (soft agar colony assay), migration (scratch wound healing assay), and invasion (Transwell assay) in vitro and decreased tumor growth and metastasis using orthotopic xenograft in vivo models. Despite strong evidence showing UHRF1 as a promising therapeutic target in several cancers, there is a current lack of UHRF1-specific inhibitors. To overcome this limitation, we explored targeting Ubiquitin-Specific Protease 7 (USP7), a deubiquitinase that prevents UHRF1 degradation, as a therapeutic strategy to trigger UHRF1 degradation. Here, we demonstrate that genetic inactivation or pharmacological inhibition of USP7 decreases TNBC tumor growth and metastasis in a UHRF1-degradation dependent manner. Phenocopying the effects of knocking out UHRF1, genetic ablation of USP7 using CRISPR/Cas9 in TNBC cell lines also resulted in significant decreases in cell clonogenicity, migration, and invasion in vitro. Intriguingly, mice bearing USP7 knockout tumors resulted in a far more robust decrease in tumor growth and absolute absence of metastatic disease. We established that many of the effects driven by USP7 ablation were mediated by the targeted degradation of UHRF1 by comparing vector control cell lines with UHRF1 knockout cells treated with the highly selective USP7 inhibitors, XL177A or FT671. In the absence of UHRF1, USP7 inhibitors showed no effects in cell migration and invasion in the three TNBC cell lines used in this study. Of promise, NSG mice bearing MDA-MB-231 tumors were recruited into vehicle or 50 mg/kg FT671 treatment groups and dosed daily via oral gavage for 20 days. For mice treated with FT671, we observed a significant decrease in tumor growth, with decreased UHRF1 levels in tumors, with minimal systemic toxicities. This study suggests that USP7 is a target with potential clinical relevance in suppressing tumor growth and metastasis in TNBC, and possibly other TP53-null cancers. These are promising results that present a novel opportunity for future clinical interventions for TNBC patients. Citation Format: Ahhyun Kim, Claudia Benavente. USP7 inhibitors prevent triple negative breast cancer metastasis by inducing UHRF1 protein degradation [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr PR04.

The Use of rAAV2-RB1-Mediated Gene Therapy in Retinoblastoma.

Retinoblastoma (RB) is a life-threatening malignancy that arises from the retina and is activated upon homozygous inactivation of the tumor suppressor RB1. Gene therapy targeting RB1 is an effective strategy to treat RB. However, it is difficult to target the RB1 gene by site-specific repair, with up to 3366 gene mutation sites identified in RB1. Thus, it is necessary to construct a promising and efficacious gene therapeutic strategy for patients with RB. To recover the function of the RB1 protein, we constructed a recombinant adeno-associated virus 2 (rAAV2) expressing RB1 that can restore RB1 function and significantly inhibit RB progression. To confirm the clinical feasibility of rAAV2-RB1, the RB1 protein was validated in vitro and in vivo after transfection. To further evaluate the clinical efficacy, RB patient-derived xenograft models were established and applied. The biosafety of rAAV2-RB1 was also validated in immunocompetent mice. rAAV2-RB1 was a rAAV2 expressing the RB1 protein, which was validated in vitro and in vivo. In vitro, rAAV2-RB1 was effectively expressed in patient-derived RB cells. In mice, intravitreal administration of rAAV2-RB1 in a population-based patient-derived xenograft trial induced limited tumor growth. Moreover, after transfection of rAAV2-RB1 in immunocompetent mice, rAAV2-RB1 did not replicate and was expressed in other important organs, except retinas, inducing minor local side effects. Our study suggested a promising efficacy gene therapeutic strategy, which might provide a chemotherapy-independent treatment option for RB.

Whole-exome sequencing explored mechanism of selpercatinib resistance in RET-rearranged lung adenocarcinoma transformation into small-cell lung cancer: a case report

Small cell transformation was one mechanism by which EGFR-mutation NSCLC acquired resistance after tyrosine kinase inhibitors (TKIs) treatment. A few reports of small cell transformation occurred in other oncogene-driven lung cancers. We found the first case of transformation of a RET-rearranged lung adenocarcinoma to SCLC after selpercatinib, a novel highly selective RET TKIs. Whole-exome sequencing (WES) was used to explore alteration in gene expression in tumor tissue at initial diagnosis and after transformation into small cell carcinoma. We found that transformed into SCLC tumor tissue had inactivation of RB1 and TP53, with RET fusion was still present. In addition, the APOBEC family of cytidine deaminases appeared amplification. Although RET rearrangement still existed, using another RET TKIs was ineffective, and etoposide plus platinum might be an effective rescue treatment.

Clonality Analysis for the Relationship between the Pulmonary Combined Neuroendocrine Carcinoma and “the So-Called Reported Histologic Transformation”

Histologic transformation (HT) is common following targeted therapy in adenocarcinoma. However, whether the transformed tumor is a new component or a combined neuroendocrine carcinoma (C-NEC) remains controversial. We aimed to explore the relationship between pulmonary C-NEC and HT. Macro-dissection was performed on different components of surgically resected C-NEC samples. Molecular alterations and clonal evolution were analyzed using whole exome sequencing (WES). The gene statuses for TP53 and RB1 were determined using immunohistochemistry (IHC) and WES to analyze the relationship between C-NEC and reported HT. Sixteen combined small-cell lung cancer patients and five combined large-cell neuroendocrine carcinoma patients were enrolled. The frequency of p53 and Rb inactivation, assessed using IHC in NEC and non-NEC components, was 76.2/76.2% and 66.7/61.9%, respectively. The expression consistency between the components was 81.0 and 85.7% for p53 and Rb, respectively. The frequencies of TP53, RB1, and EGFR mutations, assessed using WES in NEC and non-NEC components, were 81.0/81.0%, 28.6/28.6%, and 42.9/42.9%, respectively. The concordance rates for TP53, RB1, and EGFR were 90.5, 71.4, and 90.5%, respectively. The consistency rate between IHC and WES was 81.0 and 61.9% for TP53 and RB1, respectively. The different components had a common clonal origin for the 21 C-NECs in the clonal analysis, consistent with previous studies on HT. Our study shows that IHC is more sensitive for Rb detection and C-NEC, and the reported HT may be due to differences in evaluations between pathologist and clinicians. Assessing the p53/Rb and EGFR status for such cases would help in recognizing potential transformation cases or uncovering potential combined components.