Loss Of Rb Research Articles

Survey of NF1 inactivation by surrogate immunohistochemistry in ovarian carcinomas

ObjectiveInhibition of the MAPK pathway by MEK inhibitors (MEKi) is currently a therapeutic standard in several cancer types, including ovarian low-grade serous carcinoma (LGSC). A common MAPK pathway alteration in tubo-ovarian high-grade serous carcinoma (HGSC) is the genomic inactivation of neurofibromin 1 (NF1). The primary objectives of our study were to survey the prevalence of NF1 inactivation in the principal ovarian carcinoma histotype as well as to evaluate its associations with clinico-pathological parameters and key biomarkers including BRCA1/2 status in HGSC. MethodsA recently commercialized NF1 antibody (clone NFC) was orthogonally validated on an automated immunohistochemistry (IHC) platform and IHC was performed on tissue microarrays containing 2140 ovarian carcinoma cases. Expression was interpreted as loss/inactivated (complete or subclonal) versus normal/retained. ResultsLoss of NF1 expression was detected in 250/1429 (17.4%) HGSC including 11% with subclonal loss. Survival of NF1-inactivated HGSC patients was intermediate between favorable BRCA1/2 mutated HGSC and unfavorable CCNE1 high-level amplified HGSC. NF1 inactivation was mutually exclusive with CCNE1 high-level amplifications, co-occurred with RB1 loss and occurred at similar frequencies in BRCA1/2 mutated versus wild-type HGSC. NF1 loss was found in 21/286 (7.3%) endometrioid carcinomas with a favorable prognostic association (p = 0.048), and in 4/64 (5.9%) LGSC, mutually exclusive with other driver events. ConclusionsNF1 inactivation occurs in a significant subset of BRCA1/2 wild-type HGSC and a subset of LGSC. While the functional effects of NF1 inactivation need to be further characterized, this signifies a potential therapeutic opportunity to explore targeting NF1 inactivation in these tumors.

THU543 Acquired Resistance To CDK4/6 Inhibitors Is Associated With Upregulated IGF1R And IR Signaling

Abstract Disclosure: S.L. Gerhardt: None. C. Baar: None. I. Johnson: None. D. Sachdev: None. The most common subtype of breast cancer is hormone receptor-positive (HR+). While these patients are successfully treated with endocrine therapies (ET), over 30% of patients develop resistance and require other therapies. Cyclin-dependent kinases (CDKs) 4 and 6 are required for cell cycle progression. Three CDK4/6 inhibitors (CDK4/6i), palbociclib, abemaciclib, and ribociclib, have recently been approved in combination with ET. CDK4/6i block phosphorylation of retinoblastoma (Rb) and cause G1/G0 arrest and are standard of care for treating metastatic HR+ Her2- advanced breast cancer with ET. Patients who respond to the CDK4/6i eventually develop resistance to the drugs. Thus, understanding the mechanisms of resistance to CDK4/6i and the role of growth-factor signaling will allow the identification of other pathways that can be targeted. To understand the mechanisms of resistance, we generated HR+ breast cancer cells (MCF-7) with acquired resistance to palbociclib (MCF-7/PalboR) or abemaciclib (MCF-7/AbemaR) by culturing them in increasing concentrations of the drugs. As shown by others, we found that acquired resistance to CDK4/6i results in loss of Rb and thus Rb phosphorylation. We analyzed signaling pathways and in vitro proliferation in the MCF-7/PalboR and MCF-7/AbemaR compared to the CDK4/6i sensitive matched parent cells (MCF-7L MP). MCF-7/PalboR and MCF-7/AbemaR had increased levels of CDK2 and cyclin E2. Both MCF-7/PalboR and MCF-7/AbemaR cells had upregulated type I IGF receptor (IGF1R) and insulin receptor (IR) protein levels compared MCF-7L MP as assayed by western blotting and flow cytometry. MCF-7/PalboR and MCF-7/AbemaR showed higher transcript levels of the fetal form of IR (IR-A) which is not expressed in normal adult tissues. IR-A plays a role in cancer progression and has been implicated in resistance to ET. MCF-7/PalboR and MCF-7/AbemaR had increased sensitivity to signaling by insulin-like growth factor I (IGF-I) and insulin compared to MCF-7 MP, with much lower concentrations of IGF-I and insulin phosphorylating the receptors and activating downstream MAPK and PI3K/Akt/mTOR pathways. Further, IGF-I and insulin mediated growth was enhanced to a statistically greater extent in MCF-7/PalboR and MCF-7/AbemaR compared to MCF-7 MP, and this enhanced growth was not blocked by 1200 nM of palbociclib or abemaciclib. MCF-7/PalboR cells were cross resistant to abemaciclib and MCF-7/AbemaR to palbociclib. Combined treatment with CDK4/6i and rapamycin, an mTOR pathway inhibitor, was not sufficient to overcome growth-factor mediated proliferation in MCF-7/PalboR and MCF-7/AbemaR but was in MCF-7 MP. These data indicate that acquired resistance to CDK4/6i is mediated through upregulated IGF1R and IR signaling. Our data suggest that combining CDK4/6i with drugs targeting IGF1R, IR-A, and mTOR signaling should be tested to delay resistance to CDK4/6i. Presentation: Thursday, June 15, 2023

RB loss sensitizes cells to replication-associated DNA damage after PARP inhibition by trapping.

The retinoblastoma tumor suppressor protein (RB) interacts physically and functionally with a number of epigenetic modifying enzymes to control transcriptional regulation, respond to replication stress, promote DNA damage response and repair, and regulate genome stability. To better understand how disruption of RB function impacts epigenetic regulation of genome stability and determine whether such changes represent exploitable weaknesses of RB-deficient cancer cells, we performed an imaging-based screen to identify epigenetic inhibitors that promote DNA damage and compromise the viability of RB-deficient cells. We found that loss of RB alone leads to high levels of replication-dependent poly-ADP ribosylation (PARylation) and that preventing PARylation by trapping PARP enzymes on chromatin enables RB-deficient cells to progress to mitosis with unresolved replication stress. These defects contribute to high levels of DNA damage and compromised cell viability. We demonstrate this sensitivity is conserved across a panel of drugs that target both PARP1 and PARP2 and can be suppressed by reexpression of the RB protein. Together, these data indicate that drugs that target PARP1 and PARP2 may be clinically relevant for RB-deficient cancers.

RB1 loss in the mesenchymal component of onychomatricoma.

Onychomatricoma is a nail neoplasm that usually presents as longitudinal nail plate thickening, involving either the partial or whole nail. Histopathologically, it is characterized by deep invaginations of the proliferating nail matrix and proliferation of CD34+ and CD10+ spindle cells with collagenous to myxoid stroma. Onychomatricoma has been considered a fibroepithelial neoplasm. Recently, RB1 loss has been verified using array comparative genomic hybridization. This study investigated the RB1 status in onychomatricoma with morphological methods. Six patients with onychomatricoma were included in the study. RB1 status was assessed using immunohistochemical staining and fluorescence in situ hybridization. Immunohistochemical staining showed that all six cases experienced RB1 loss in the mesenchymal component of onychomatricoma but not in the proliferated nail matrix. Fluorescence in situ hybridization in five cases showed a monoallelic deletion of the RB1 locus in the mesenchymal component but not in the proliferated nail matrix. RB1 loss was observed only in the mesenchymal component of onychomatricoma. Our findings suggest that the proliferated nail matrix in onychomatricoma represents reactive hyperplasia of various degrees secondary to neoplastic mesenchymal proliferation. This indicates that onychomatricoma should be recognized as an RB1-deleted soft tissue neoplasm rather than a fibroepithelial neoplasm.

Complete loss of TP53 and RB1 is associated with complex genome and low immune infiltrate in pleomorphic rhabdomyosarcoma

Rhabdomyosarcoma accounts for roughly 1% of adult sarcomas, with pleomorphic rhabdomyosarcoma (PRMS) as the most common subtype. Survival outcomes remain poor for patients with PRMS, and little is known about the molecular drivers of this disease. To better characterize PRMS, we performed a broad array of genomic and immunostaining analyses on 25 patient samples. In terms of gene expression and methylation, PRMS clustered more closely with other complex karyotype sarcomas than with pediatric alveolar and embryonal rhabdomyosarcoma. Immune infiltrate levels in PRMS were among the highest observed in multiple sarcoma types and contrasted with low levels in other rhabdomyosarcoma subtypes. Lower immune infiltrate was associated with complete loss of both TP53 and RB1. This comprehensive characterization of the genetic, epigenetic, and immune landscape of PRMS provides a roadmap for improved prognostications and therapeutic exploration.

Lineage plasticity and treatment resistance in prostate cancer: the intersection of genetics, epigenetics, and evolution.

Androgen deprivation therapy is a cornerstone of treatment for advanced prostate cancer, and the development of castrate-resistant prostate cancer (CRPC) is the primary cause of prostate cancer-related mortality. While CRPC typically develops through a gain in androgen receptor (AR) signaling, a subset of CRPC will lose reliance on the AR. This process involves genetic, epigenetic, and hormonal changes that promote cellular plasticity, leading to AR-indifferent disease, with neuroendocrine prostate cancer (NEPC) being the quintessential example. NEPC is enriched following treatment with second-generation anti-androgens and exhibits resistance to endocrine therapy. Loss of RB1, TP53, and PTEN expression and MYCN and AURKA amplification appear to be key drivers for NEPC differentiation. Epigenetic modifications also play an important role in the transition to a neuroendocrine phenotype. DNA methylation of specific gene promoters can regulate lineage commitment and differentiation. Histone methylation can suppress AR expression and promote neuroendocrine-specific gene expression. Emerging data suggest that EZH2 is a key regulator of this epigenetic rewiring. Several mechanisms drive AR-dependent castration resistance, notably AR splice variant expression, expression of the adrenal-permissive 3βHSD1 allele, and glucocorticoid receptor expression. Aberrant epigenetic regulation also promotes radioresistance by altering the expression of DNA repair- and cell cycle-related genes. Novel therapies are currently being developed to target these diverse genetic, epigenetic, and hormonal mechanisms promoting lineage plasticity-driven NEPC.

Lipoblastoma-Like Tumor and Fibrosarcoma-Like Lipomatous Neoplasm Represent the Same Entity: A Clinicopathologic and Molecular Genetic Study of 23 Cases Occurring in Both Men and Women at Diverse Locations

Lipoblastoma-like tumor (LLT) is a benign soft tissue tumor demonstrating mixed morphologic features of lipoblastoma, myxoid liposarcoma, and spindle cell lipoma but lacking genetic alterations associated with those tumors. LLT was originally thought to be specific to the vulva but has since been reported in the paratesticular region. The morphologic features of LLT overlap with those of “fibrosarcoma-like lipomatous neoplasm” (FLLN), a rare, indolent adipocytic neoplasm considered by some to form part of the spectrum of atypical spindle cell and pleomorphic lipomatous tumor. We compared the morphologic, immunohistochemical, and genetic features of 23 tumors previously classified as LLT (n = 17) and FLLN (n = 6). The 23 tumors occurred in 13 women and 10 men (mean age, 42 years; range, 17 to 80 years). Eighteen (78%) cases arose in the inguinogenital region, whereas 5 tumors (22%) involved noninguinogenital soft tissue, including the flank (n = 1), shoulder (n = 1), foot (n = 1), forearm (n = 1), and chest wall (n = 1). Microscopically, the tumors were lobulated and septated, with variably collagenized fibromyxoid stroma, prominent thin-walled vessels, scattered univacuolated or bivacuolated lipoblasts, and a minor component of mature adipose tissue. Using immunohistochemistry, 5 tumors (42%) showed complete RB1 loss, with partial loss in 7 cases (58%). RNA sequencing, chromosomal microarray, and DNA next-generation sequencing study results were negative for significant alterations. There were no clinical, morphologic, immunohistochemical, or molecular genetic differences between cases previously classified as LLT or FLLN. Clinical follow-up (11 patients [48%]; range, 2-276 months; mean, 48.2 months) showed all patients were alive without disease, and only one patient had experienced a single local recurrence. We conclude that LLT and FLLN represent the same entity, for which “LLT” seems most appropriate. LLT may occur in either sex and any superficial soft tissue location. Careful morphologic study and appropriate ancillary testing should allow for the distinction of LLT from its potential mimics.

Etoposide/platinum plus anlotinib for patients with transformed small-cell lung cancer from EGFR-mutant lung adenocarcinoma after EGFR-TKI resistance: a retrospective and observational study.

The histological conversion of lung adenocarcinoma (LUAD) into small-cell lung cancer (SCLC) is an important resistance mechanism for epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI)-resistant LUAD. Anlotinib has been recommended as the third-line treatment for SCLC patients. The efficacy of etoposide/platinum (EP) as the main treatment is very limited for patients with transformed SCLC. However, little is known about EP plus anlotinib for transformed SCLC. The present study retrospectively explored the clinical response to EP combined with anlotinib in patients with transformed SCLC from LUAD after EGFR-TKI failure. A total of 10 patients who underwent SCLC transformation from EGFR-TKI-resistant LUAD were retrospectively reviewed from September 1, 2019, to December 31, 2022, in three regional hospitals. All of the patients were treated with the combination regimen of EP and anlotinib for four to six cycles, followed by anlotinib maintenance therapy. The clinical efficacy indices including objective response rate (ORR), disease control rate (DCR), median progression-free survival (mPFS), median overall survival (mOS), and toxicities were evaluated. The median time from EGFR-TKI treatment to SCLC conversion was 20.1 ± 2.76 months (17-24 months). Genetic examination after transformation showed that 90% of the patients retained their original EGFR gene mutations. Additional driver genes were found, including BRAF mutation (10%), PIK3CA mutation (20%), RB1 loss (50%), and TP53 mutation (60%). The ORR and DCR were 80% and 100%, respectively. The mPFS was 9.0 months (95% CI, 7.9-10.1 months), and the mOS was 14.0 months (95% CI, 12.0-15.9 months). Less than 10% of grade 3 toxicities were observed, and no grade 4 toxicity and death events were reported. The EP plus anlotinib regimen appears to be a promising and safe strategy in transformed SCLC patients after EGFR-TKI resistance, which warrants further investigation.

Abstract B063: Interplay between PARP and the RB/E2F axis in prostate cancer

Abstract Retinoblastoma (RB) protein is a tumor suppressor that represses the transcriptional activity of E2Fs by forming an RB-E2F repressor complex. The phosphorylation of RB by CDK4/6 results in dissociation of the complex and subsequent E2F transcriptional activity, leading to cell cycle progression. RB loss occurs in 10-15% of castration-resistant prostate cancer (CRPC) which is associated tumor aggressiveness and poor clinical outcomes. RB loss is also associated differential treatment response to an array of treatment modalities, including hormone therapy and DNA damage inducing chemotherapy. RB depletion leads to enhanced expression of DNA repair proteins due to increased transcriptional activity of E2F1. PARP-1 is an enzyme that plays a role in multiple nuclear processes including DNA repair, transcriptional regulation, and chromatin dynamics. Increased PARP-1 activity is associated with and correlates to poor clinical outcomes in CRPC. E2F1 transcriptional activity is supported by PARP enzymatic activity, but the consequences of the functional interaction between the RB/E2F axis and PARP remains an open line of inquiry, which may yield novel treatment strategies and/or biomarkers of response. Isogenic models of RB1 knockdown were used to evaluate the impact of RB loss on PARP enzymatic activity. PARP-1 protein increases upon RB1 depletion with a concomitant increase in PARP activity. Growth curve assays were conducted to examine the biological response to PARP inhibition in the isogenic models. Results suggest a differential response between control and RB1 depleted cells when treated with Olaparib. RB1depleted cells also exhibit differential PARP activity in response to treatment with the IC50 dose of Olaparib. Future studies have been designed to evaluate the impact of RB alteration and manipulation of PARP activity on biological and molecular processes that are governed by RB/E2F and PARP. Citation Format: Latese Evans, Moriah Cunningham, Jasibel Vasquez Gonzalez, Matthew Schiewer. Interplay between PARP and the RB/E2F axis in prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B063.

Abstract PR004: Exploring the role of ASCL1 in neuroendocrine prostate cancer

Abstract Most patients with prostate adenocarcinoma (PAd), an androgen receptor (AR) driven cancer, develop resistance to therapies targeting AR. Consequently, a portion of these patients develop neuroendocrine prostate cancer (NEPC), a rapidly progressing cancer with limited therapies and poor survival outcomes. Current research to understand the transition of PAd to NEPC suggests a model of lineage plasticity, where AR-dependent luminal tumors progress towards an AR-independent neuroendocrine lineage. Several groups have shown human NEPC tumors have lost RB1 and TP53, and in experimental models, loss of both genes mediates the transition to a neuroendocrine lineage. Notably, NEPC histology and gene expression resemble another neuroendocrine cancer, small cell lung carcinoma (SCLC), also characterized by loss of RB1 and TP53. In SCLC, transcription factor ASCL1 is required for tumor cell growth in vitro and for SCLC formation in vivo. ASCL1 is also present in NEPC tumors and in NEPC cell line models. In fact, ASCL1 was shown to regulate neuronal stem cell-like lineage programming in NEPC in vitro. We aimed to determine if ASCL1 is required in the transition of PAd to NEPC in vivo and to define its function in NEPC. To model the PAd to NEPC transition, we established genetically engineered mouse models (GEMMs) harboring loss of RB1 and TP53 with MYC overexpression (RPM) by administering adenovirus expressing Cre recombinase directly to the prostate of these GEMMs. These animals display prostate tumors with small cell histology and are heterogeneous for neuroendocrine markers such as ASCL1, NEUROD1, and INSM1. We recently determined that the concomitant loss of ASCL1 in this model (RPMA) does not stop the formation of tumors with small cell histology. Notably, a subset of the cells with small cell histology are NEUROD1+, with a higher proportion of NEUROD1+ cells present compared to the RPM model with intact ASCL1, suggesting the possibility that NEUROD1 in the same or distinct cells is compensating for the absence of ASCL1. In a separate paradigm using these same GEMMs to establish prostate organoids, we show their capacity to generate subcutaneous allograft tumors displaying mixed histology including neuroendocrine small cell features expressing ASCL1, NEUROD1, and INSM1. Strikingly, subcutaneous allografts from prostate organoids lacking ASCL1 exhibit a dramatic reduction in tumor formation efficiency, lack small cell histology including NEUROD1 and INSM1 and other neuroendocrine markers, and often result in the development of cysts rather than tumors. These results demonstrate complex functions for ASCL1 in neuroendocrine lineage programming while suggesting additional functions in supporting prostate tumor formation. Citation Format: Kathia E. Rodarte, Lydia Flores, Vishal Kandagatla, Juan Villarreal, Trisha K. Savage, Su Deng, Ping Mu, Rajal B. Shah, Trudy G. Oliver, Jane E. Johnson. Exploring the role of ASCL1 in neuroendocrine prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr PR004.

Abstract B012: Genomic and epigenomic drivers of double-negative metastatic prostate cancer

Abstract Systemic targeted therapy in prostate cancer is primarily focused on ablating androgen receptor (AR) signaling. Androgen deprivation therapy and second-generation AR-targeted therapy selectively favor the development of treatment-resistant subtypes of metastatic castration-resistant prostate cancer (mCRPC), defined by whether the tumor expresses either AR or neuroendocrine (NE) markers. Among the subtypes of mCRPC, the molecular drivers of double-negative (AR-/NE-) mCRPC are poorly defined. In this study, we comprehensively characterize genomic and epigenomic features of treatment-emergent mCRPC subtypes in 210 tumors by integrating matched RNA sequencing, whole-genome sequencing, and whole-genome bisulfite sequencing. We show that AR-/NE- tumors exhibit a clinically and molecularly distinct phenotype. Patients with AR-/NE- mCRPC tumors have the shortest survival, and these tumors preferentially harbor amplification of the chromatin remodeler CHD7 and loss of PTEN. We demonstrate that methylation changes in CHD7 candidate enhancers are linked to elevated CHD7 expression in AR-/NE+ tumors. Moreover, we use genome-wide methylation analysis to nominate the Krüppel-like factor gene KLF5 as a driver of the AR-/NE- phenotype and link its activity to loss of the tumor suppressor RB1. These observations reveal the aggressiveness of the AR-/NE- tumors and elucidate genomic and epigenomic drivers of mCRPC subtypes, which may facilitate the identification of novel therapeutic targets in this highly aggressive disease. Citation Format: Arian Lundberg, Meng Zhang, Rahul R. Aggarwal, Haolong Li, Li Zhang, Adam Foye, Martin Sjöström, Jonathan Chou, Kevin Chang, Thaidy Moreno-Rodriguez, Raunak Shrestha, Avi Baskin, Xiaolin Zhu, Alana S. Weinstein, Noah Younger, Joshi J. Alumkal, Tomasz M. Beer, Kim N. Chi, Christopher P. Evans, Martin Gleave, Primo N. Lara, Robert E. Reiter, Matthew B. Rettig, Owen N. Witte, Alexander W. Wyatt, Felix Y. Feng, Eric J. Small, David A. Quigley. Genomic and epigenomic drivers of double-negative metastatic prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B012.

Abstract B015: HOXA9 promotes enzalutamide resistance in RB-p53 deficient prostate cancer

Abstract Introduction: Castration resistant prostate cancer (CRPC) cells can acquire resistance to the androgen receptor (AR) inhibitor enzalutamide (EZ). These cells can switch lineages from an adenocarcinoma to a neuroendocrine (NE) cell type that proliferate independently of the AR signaling pathway. Cancer genomic and molecular studies identified that co-deletion of the retinoblastoma (RB1) and TP53 genes can promote the acquisition of EZ resistance and neuroendocrine features. However, RB and p53 are both tumour suppressor and are therefore difficult to target pharmacologically. The purpose of this study is to identify an actionable molecular factor downstream of RB and p53 that drives EZ resistance in CRPC. Methods and Results: To characterize functional and molecular features of RB and p53 loss, CRISPR-Cas9 was used to generate a double knockout (DKO) line in LNCaP prostate cancer cells. Compared with LNCaP wild type (WT) cells, only DKOs formed distinct colonies over a 4-week colony forming assay under EZ treatment. RNA sequencing of DKO and WT cells, followed by gene ontology (GO) analysis, revealed NE and stemness genes, including HOXA9, were significantly upregulated in DKO cells. To categorize gene loss events in EZ-treated LNCaP cells, a genome-wide CRISPR knockout screen was performed. Pools of KO cells were treated with either EZ or DMSO and then analyzed by next generation sequencing to identify gene mutations that confer increased resistance or sensitivity to EZ. GO analysis of de-enriched genes following EZ treatment identified stemness genes, including HOXA9, highlighting the potential importance of a stem-like phenotype for acquiring EZ resistance EZ. To further investigate the functional significance of HOXA9 we analyzed EZ-resistant prostate tumour genomic data. HOXA9 is mutated or mis-expressed in 10% of cases. Importantly, HOXA9 is either amplified or overexpressed in virtually all these cases, and is associated with poorer prognosis, suggesting an oncogenic role for HOXA9 in CRPC. HOXA9 transcript levels were positively correlated with neuroendocrine features and negatively correlated with RB1expression in these tumour samples. LNCaP WT and DKO cells we then engineered to overexpress HOXA9 displayed increased IC50 values following a 6-day EZ treatment, compared with either parental line. DKO cells that overexpress HOXA9 also formed significantly more colonies following a 4-week EZ treatment, compared with parentals. In contrast, shRNA knockdown of HOXA9 caused a reduction in IC50 values and formed fewer drug resistant colonies compared with control cells. Finally, DKO and WT cells were co-treated with varying concentrations of the HOXA9 inhibitor DB818 and EZ. DKO cells were more sensitive to DB818 alone at high concentrations and displayed higher synergy as measured by a ZIP synergy score when co-treated with EZ. Conclusions: Overall, these results suggest that HOXA9 regulates EZ resistance in prostate cancer. Furthermore, HOXA9 inhibition may be of therapeutic benefit for treating EZ-resistant CRPC. Citation Format: Michael V. Roes, Fred A. Dick. HOXA9 promotes enzalutamide resistance in RB-p53 deficient prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B015.

Biomarker testing and treatment patterns in US patients (pts) with advanced/metastatic non-small cell lung cancer (NSCLC) harboring MET amplification.

e21057 Background: Limited published evidence exists on pts with advanced stage NSCLC harboring MET amplification ( METamp), a rare oncogenic driver. We report here real-world biomarker testing and treatment patterns for these patients. Methods: This was a non-interventional, descriptive cohort study using ConcertAI electronic medical record data derived from US community oncology centers and linked with ASCO/CancerLinQ data. Eligible pts had stage IIIB–IV NSCLC and METamp. Study period: Jan 1, 2004 – Oct 31, 2021. Results: Of 8,454 lung cancer pts with minimum 1 molecular test including MET, 164 had METamp (1.9%). Pts were mainly identified from 2015 onwards and primarily in oncology in the Southern and Midwest regions (86.6%). Majority of pts were tested by tissue biopsy (67.1%), 20.7% by liquid biopsy and 12.2% not specified. At least 64.5% of pts were highly MET amplified (either gene copy number ≥8 or by liquid biopsy ++, +++). Other mutations screened for were ALK, ROS1 and EGFR (99.4%, 98.2% and 97.6%), with oncogenic ALK, ROS1 fusions and activating EGFR mutations in 0.6% and 13.1%, respectively. BRAF and KRAS were less frequently screened for (90.2% and 87.2%), with potential oncogenic alterations in 2.0% and 12.6%. Of 127 pts tested for PD-L1 (by immunohistochemistry), 63.0% were positive, and 72.5% had tumor proportion score ≥50. Eighteen pts had METamp detected after receiving an EGFR tyrosine kinase inhibitor (TKI), indicating METamp as a secondary oncogenic driver. RB1 loss and MYC amplification, as potential mechanisms of MET inhibition resistance, were not frequently screened for (30.5% and 46.3%). RB1 loss was identified in 2.0% and MYC amplification in 15.8%. Next generation sequencing was the most frequent diagnostic used overall. A total of 144 pts (87.8% of 164 pts) received first-line (1L) anticancer therapy; platinum-based chemotherapy 57/144 (39.6%), immune-checkpoint inhibitors 50/144 (34.7%) or EGFR TKI monotherapy 24/144 (16.7%), other 2/144 (1.4%) and MET TKIs (mainly crizotinib) were used in 11/144 pts (7.6%) in 1L, 14/78 (17.9) in second line and 7/42 (16.7%) in third line. Conclusions: In this cohort of pts identified in a US community oncology setting, biomarker screening even for rarer genetic alterations occurred. Concomitant genetic alterations to METamp were infrequent except for alterations of EGFR and KRAS. The frequency of pts with high level of METamp in the cohort may explain the lower frequency of other biomarkers, as high level of amplification indicates a cleaner MET profile. Overall MET inhibitor treatment was not common. Screening for novel and rare biomarkers such as METamp as a primary or secondary oncogenic driver is becoming increasingly important to enable efficacious targeted treatment.

Circulating tumor cells (CTCs) in patients with metastatic castration resistant prostate cancer (mCRPC) treated with olaparib plus 177lutetium-prostate specific membrane antigen-617 (177Lu-PSMA-617) in the LuPARP trial.

5064 Background: LuPARP (NCT03874884) is evaluating the safety and efficacy of olaparib in combination with 177Lu-PSMA-617 in patients (pts) with mCRPC who have progressed on an androgen receptor pathway inhibitors and docetaxel. We aim to identify potential prognostic and predictive biomarkers from serial CTC profiling. Methods: Twenty-six pts with high PSMA expression on PSMA-PET received 7.4 GBq of 177Lu-PSMA-617 every 6 weeks (wk) together with an escalating dose-schedule of olaparib (50mg BD – 300mg BD, days 2 to 14 or days -4 to 14) for up to 6 cycles. For CTC analysis, 10 mls of blood was collected at baseline, 12-weekly for 48 wk, and thereafter every 24 wk and at disease progression. CK+, CD45-, and DAPI+ CTCs were enumerated from 3 mls of blood and immunoassayed for PSMA expression using the Epic Sciences platform. Correlations between total and PSMA+ CTC counts and PSA50 response (PSA reduction of ≥ 50%) were evaluated. Low pass whole genome sequencing (lpWGS) was performed on baseline and longitudinal CTC samples to identify copy number alterations. Results: At baseline, 23 of 26 pts (88%) had detectable CTCs (median 2.9 CTC/ml, range 0-30), and of these 23 pts, 17 (74%) had detectable PSMA+ CTCs (median 0.9 CTC/ml, range 0.3-27), indicating heterogeneity of PSMA expression in CTCs. The CTC positivity rates (% of cases ≥1 CTC) at week (w)12, w24, w36, w48 and at disease progression were 57%, 58%, 75%, 36% and 100%, and the PSMA positivity rates (% of cases ≥1 PSMA+ CTC) were 38%, 36%, 22%, 50% and 56%, respectively. Fifteen of 20 (75%) evaluable pts with paired baseline and w12 CTCs had a ≥50% decline in total CTC count, of which 9/15 (60%) pts also achieved a PSA50 response. Five of these 9 (56%) pts had 100% CTC clearance at w12. In terms of PSMA+ CTCs, 14/15 (93%) evaluable pts had ≥50% PSMA+ CTC decline with 13 pts (93%) achieving complete CTC clearance by w12. Six of the 13 pts with complete PSMA+ CTC clearance at w12 also achieved a PSA50 response. Beyond PSMA expression, genomic heterogeneity was evident in baseline and on-treatment CTCs, including recurrent loss of PTEN, TP53, BRCA2, ATM, and RB1, and gain of AR and MYC. lpWGS in 10 baseline samples identified 5 cases with BRCA2 loss and 5 cases with ATM loss. Two pts with BRCA2 loss and 3 pts with ATM loss had a PSA50 response by w12. Conclusions: We observed high rates of total and PSMA+ CTCs in PSMA-expressing mCRPC. Total and PSMA+ CTCs decline with combined olaparib and 177Lu-PSMA-617 treatment. Early declines in total and PSMA+ CTCs including 12w CTC clearance paralleled PSA responses. Notably, total and PSMA+ CTCs rise again in the setting of disease progression. Serial CTC profiling may assist in tracking response to 177Lu-PSMA-617 therapy. Clinical trial information: NCT03874884 .

Ferroptosis: the vulnerability within a cancer monster.

Treatment-resistant cancer, such as neuroendocrine prostate cancer (NEPC), is a lethal disease with limited therapeutic options. RB1 is a tumor suppressor gene that is lost in a majority of NEPC tumors. In this issue of the JCI, Wang and colleagues examined how RB1 loss may sensitize cancer cells to ferroptosis inducers through elevation of ACSL4, a key enzyme that promotes lipid peroxidation and triggers ferroptosis. We discuss a high potential of RB1-deficient cells to undergo ferroptosis due to the elevation of ACSL4. This is normally kept in check by abundant expression of GPX4, an antioxidant enzyme, in cancer cells. This balance, however, is tilted by GPX4 inhibitors, leading to massive ferroptosis. We highlight possible therapeutic strategies that exploit this inherent vulnerability for targeting RB1-deficient, treatment-resistant cancer.

DNA-PKcs as an upstream mediator of OCT4-induced MYC activation in small cell lung cancer

Small cell lung cancer (SCLC) is a neuroendocrine tumor noted for the rapid development of both metastases and resistance to chemotherapy. High mutation burden, ubiquitous loss of TP53 and RB1, and a mutually exclusive amplification of MYC gene family members contribute to genomic instability and make the development of new targeted agents a challenge. Previously, we reported a novel OCT4-induced MYC transcriptional activation pathway involving c-MYC, pOCT4S111, and MAPKAPK2 in progressive neuroblastoma, also a neuroendocrine tumor. Using tumor microarray analysis of clinical samples and preclinical models, we now report a correlation in expression between these proteins in SCLC. In correlating c-MYC protein expression with genomic amplification, we determined that some SCLC cell lines exhibited high c-MYC without genomic amplification, implying amplification-independent MYC activation. We then confirmed direct interaction between OCT4 and DNA-PKcs and identified specific OCT4 and DNA-PKcs binding sites. Knock-down of both POU5F1 (encoding OCT4) and PRKDC (encoding DNA-PKcs) resulted in decreased c-MYC expression. Further, we confirmed binding of OCT4 to the promoter/enhancer region of MYC. Together, these data establish the presence of a DNA-PKcs/OCT4/c-MYC pathway in SCLCs. We then disruptively targeted this pathway and demonstrated anticancer activity in SCLC cell lines and xenografts using both DNA-PKcs inhibitors and a protein-protein interaction inhibitor of DNA-PKcs and OCT4. In conclusion, we demonstrate here that DNA-PKcs can mediate high c-MYC expression in SCLCs, and that this pathway may represent a new therapeutic target for SCLCs with high c-MYC expression.

Chromophobe Renal Cell Carcinoma with Sarcomatoid Differentiation: Clinicopathologic Correlation and Molecular Findings.

Introduction: Sarcomatoid differentiation has been reported in approximately 8% of chromophobe renal cell carcinoma (RCC) and is associated with a worse prognosis. We aim to describe the clinicopathologic and molecular findings of chromophobe RCC with sarcomatoid differentiation. Methods: Surgical pathology database was searched to identify chromophobe RCC with sarcomatoid differentiation from January 2015 to December 2021. Results: Five patients were diagnosed with chromophobe RCC with sarcomatoid differentiation. The median age at the time of diagnosis was 57 years (range 51-61 years). Three patients died after median follow-up of 12.1 months (range 1.6-18.2 months). The median tumor size was 10.7 cm (range 5.6-13.6 cm). The median percentage of sarcomatoid component was 60% (range 10-90%), and the median percentage of necrosis was 30% (range 10-50%). One tumor demonstrated osteoid formation. PAX8, keratin 7, KIT (CD117), and Hale colloidal iron were positive in the epithelial component, whereas the sarcomatoid component was positive for vimentin, CD10, and high Ki67 proliferative index. Molecular testing was performed in three specimens: all were TP53 mutated and microsatellite stable. One aggressive tumor had RB1 frameshift mutation and copy number gains for TERT and CUL4A. Conclusion: Chromophobe RCC with sarcomatoid differentiation is a rare entity with aggressive behavior. Percentage of sarcomatoid component, necrosis, and the occurrence of metastasis is associated with worse prognosis. Molecular profiling reveals frequent TP53 mutation. While TERT promoter mutation has no prognostic implication, FLCN inactivation may be associated with a less aggressive course. The clinical significance of RB1 loss is unclear.

Significance of RB Loss in Unlocking Phenotypic Plasticity in Advanced Cancers.

Cancer cells can undergo plasticity in response to environmental stimuli or under selective therapeutic pressures that result in changes in phenotype. This complex phenomenon of phenotypic plasticity is now recognized as a hallmark of cancer. Lineage plasticity is often associated with loss of dependence on the original oncogenic driver and is facilitated, in part, by underlying genomic and epigenetic alterations. Understanding the molecular drivers of cancer plasticity is critical for the development of novel therapeutic strategies. The retinoblastoma gene RB1 (encoding RB) is the first tumor suppressor gene to be discovered and has a well-described role in cell-cycle regulation. RB is also involved in diverse cellular functions beyond cell cycle including differentiation. Here, we describe the emerging role of RB loss in unlocking cancer phenotypic plasticity and driving therapy resistance across cancer types. We highlight parallels in cancer with the noncanonical role of RB that is critical for normal development and lineage specification, and the downstream consequences of RB loss including epigenetic reprogramming and chromatin reorganization that can lead to changes in lineage program. Finally, we discuss potential therapeutic approaches geared toward RB loss cancers undergoing lineage reprogramming.

BET Inhibition Sensitizes Immunologically Cold Rb-Deficient Prostate Cancer to Immune Checkpoint Blockade.

Non-T-cell-inflamed immunologically "cold" tumor microenvironments (TME) are associated with poor responsiveness to immune checkpoint blockade (ICB) and can be sculpted by tumor cell genomics. Here, we evaluated how retinoblastoma (Rb) tumor-suppressor loss-of-function (LOF), one of the most frequent alterations in human cancer and associated with lineage plasticity, poor prognosis, and therapeutic outcomes, alters the TME, and whether therapeutic strategies targeting the molecular consequences of Rb loss enhance ICB efficacy. We performed bioinformatics analysis to elucidate the impact of endogenous Rb LOF on the immune TME in human primary and metastatic tumors. Next, we used isogenic murine models of Rb-deficient prostate cancer for in vitro and in vivo mechanistic studies to examine how Rb loss and bromodomain and extraterminal (BET) domain inhibition (BETi) reprograms the immune landscape, and evaluated in vivo therapeutic efficacy of BETi, singly and in combination with ICB and androgen deprivation therapy. Rb loss was enriched in non-T-cell-inflamed tumors, and Rb-deficient murine tumors demonstrated decreased immune infiltration in vivo. The BETi JQ1 increased immune infiltration into the TME through enhanced tumor cell STING/NF-κB activation and type I IFN signaling within tumor cells, resulting in differential macrophage and T-cell-mediated tumor growth inhibition and sensitization of Rb-deficient prostate cancer to ICB. BETi can reprogram the immunologically cold Rb-deficient TME via STING/NF-κB/IFN signaling to sensitize Rb-deficient prostate cancer to ICB. These data provide the mechanistic rationale to test combinations of BETi and ICB in clinical trials of Rb-deficient prostate cancer.

Abstract 1714: CRACD/KIAA1211 loss drives cell plasticity and immune evasion of small cell lung cancer

Abstract Small cell lung carcinoma (SCLC) is a lethal neuroendocrine type of lung cancer with limited therapeutic options. Despite recent advances in cancer immunotherapy, its efficacy is limited to a small subset of SCLC patient tumors. The molecular origin of the refractoriness to immunotherapy remains elusive. CRACD (Capping protein inhibiting regulator of actin dynamics; KIAA1211/CRAD) gene is frequently mutated and transcriptionally downregulated in SCLC. Cracd knockout (KO) causes the transformation of preneoplastic neuroendocrine cells and significantly accelerates SCLC development in a mouse model initiated by the loss of Rb1, Trp53, and Rbl2 in the lung epithelium. Cracd KO induces tumor cell plasticity generating deregulated cell lineage trajectories of SCLC tumors. Strikingly, Cracd KO SCLC tumors display the complete loss of CD8+ T cells due to epigenetic suppression of the MHC-I pathway. Furthermore, single-cell transcriptomic analyses of SCLC patient samples classified SCLC by concurrent features: CRACD inactivation and tumor antigen presentation impairment. This study suggests CRACD as a tumor suppressor of SCLC that regulates proliferation and immune recognition of cells, providing novel insight into the mechanism of SCLC evading immune surveillance. Citation Format: Shengzhe Zhang, Kee-Bum Kim, Yuanjian Huang, Dong-Wook Kim, Bongjun Kim, Kyung-Pil Ko, Gengyi Zou, Jie Zhang, Sohee Jun, Nicole A. Kirk, Ye Eun Hwang, Young Ho Ban, Joseph M. Chan, Charles M. Rudin, Kwon-Sik Park, Jae-Il Park. CRACD/KIAA1211 loss drives cell plasticity and immune evasion of small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1714.