Inactivation Of Rb Research Articles

Inactivation of RB1, CDKN2A, and TP53 have distinct effects on genomic stability at side-by-side comparison in karyotypically normal cells.

Chromosomal instability is a common feature in malignant tumors. Previous studies have indicated that inactivation of the classical tumor suppressor genes RB1, CDKN2A, and TP53 may contribute to chromosomal aberrations in cancer by disrupting different aspects of the cell cycle and DNA damage checkpoint machinery. We performed a side-by-side comparison of how inactivation of each of these genes affected chromosomal stability in vitro. Using CRISPR-Cas9 technology, RB1, CDKN2A, and TP53 were independently knocked out in karyotypically normal immortalized cells, after which these cells were followed over time. Bulk RNA sequencing revealed a distinct phenotype with upregulation of pathways related to cell cycle control and proliferation in all three knockouts. Surprisingly, the RB1 and CDKN2A knocked out cell lines did not harbor more copy number aberrations than wild-type cells, despite culturing for months. The TP53-knocked out cells, in contrast, showed a massive amount of copy number alterations and saltatory evolution through whole genome duplication. This side-by-side comparison indicated that the effects on chromosomal stability from inactivation of RB1 and CDKN2A are negligible compared to inactivation of TP53, under the same conditions in a nonstressful environment, even though partly overlapping regulatory pathways are affected. Our data suggest that loss of RB1 and CDKN2A alone is not enough to trigger surviving detectable aneuploid clones while inactivation of TP53 on its own caused massive CIN leading to saltatory clonal evolution in vitro and clonal selection.

Concurrent inhibition of CDK2 adds to the anti-tumour activity of CDK4/6 inhibition in GIST.

Advanced gastrointestinal stromal tumour (GIST) is characterised by genomic perturbations of key cellcycle regulators. Oncogenic activation of CDK4/6 results in RB1 inactivation and cell cycle progression. Given that single-agent CDK4/6 inhibitor therapy failed to show clinical activity in advanced GIST, we evaluated strategies for maximising response to therapeutic CDK4/6 inhibition. Targeted next-generation sequencing and multiplexed protein imaging were used to detect cellcycle regulator aberrations in GIST clinical samples. The impact of inhibitors of CDK2, CDK4 and CDK2/4/6 was determined through cellproliferation and proteindetection assays. CDK-inhibitor resistance mechanisms were characterised in GIST cell lines after long-term exposure. We identify recurrent genomic aberrations in cellcycle regulators causing co-activation of the CDK2 and CDK4/6 pathways in clinical GIST samples. Therapeutic co-targeting of CDK2 and CDK4/6 is synergistic in GIST cell lines with intact RB1, through inhibition of RB1 hyperphosphorylation and cell proliferation. Moreover, RB1 inactivation and a novel oncogenic cyclin D1 resulting from an intragenic rearrangement (CCND1::chr11.g:70025223) are mechanisms of acquired CDK-inhibitor resistance in GIST. These studies establish the biological rationale for CDK2 and CDK4/6 co-inhibition as a therapeutic strategy in patients with advanced GIST, including metastatic GIST progressing on tyrosine kinase inhibitors.

Interruption of aberrant chromatin looping is required for regenerating RB1 function and suppressing tumorigenesis

RB transcriptional corepressor 1 (RB1) is a critical regulatory gene in physiological and pathological processes. Genetic mutation is considered to be the main cause of RB1 inactivation. However, accumulating evidence has shown that not all RB1 dysfunction is triggered by gene mutations, and the additional mechanism underlying RB1 dysfunction remains unclear. Here, we firstly reveal that a CCCTC binding factor (CTCF) mediated intrachromosomal looping served as a regulatory inducer to inactivate RB1. Once the core genomic fragment was deleted by Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 (CRISPR/Cas9), this intrachromosomal looping was disrupted. After the open of chromatin, Enhancer of Zeste Homolog 2 (EZH2) was released and decreased the level of Tri-Methyl-Histone H3 Lys27 (H3K27me3) at the RB1 promoter, which substantially restored the expression of RB protein (pRB) and inhibited tumorigenesis. In addition, targeted correction of abnormal RB1 looping using the small-molecule compound GSK503 efficiently restored RB1 transcription and suppressed tumorigenesis. Our study reveals an alternative transcriptional mechanism underlying RB1 dysfunction independent of gene mutation, and advancing the discovery of potential therapeutic chemicals based on aberrant chromatin looping.

Abstract A020: Combined inhibition of SHP2 and CDK4/6 is active in preclinical models of NF1-associated malignant peripheral nerve sheath tumor

Abstract Background: NF1 is an essential negative regulator of RAS activity and its function is lost in nearly 90% of malignant peripheral nerve sheath tumors (MPNST). Additional recurrent molecular changes include loss of function alterations in CDKN2A, TP53, EED and SUZ12, but molecular targeting of these genomic events represents a unique challenge. We previously reported that the efficacy of MEK inhibitor is limited by adaptive activation of receptor tyrosine kinases and the adaptor protein SHP2, and that combined inhibition of MEK and SHP2 is effective in MPNST. However, the clinical potential of combination MEK and SHP2 inhibitors may be limited by the overlapping toxicity induced by ERK pathway inhibition. Loss of CDKN2A, inactivation of RB1, and hyperactivation of cyclin dependent kinases (CDK) in MPNST also suggest that small-molecule CDK4/6 inhibitors (CDK4/6i) may be a potential therapeutic strategy, but monotherapy with CDK4/6i also demonstrates limited activity. We hypothesize that the anti-tumor response of SHP2i may be potentiated by agents targeting the cell cycle in combination. Methods: The effects of shRNA-mediated inducible SHP2 knockdown on RAS signaling, short-term and long-term cell growth, and response to CDK4/6i were examined using immunoblotting, high throughput proliferation assays, and colony formation assays. Combined effects of SHP2i plus CDK4/6i on signaling, cell cycle, apoptosis, cell and in vivo tumor growth were assessed. Pharmacodynamic (PD) assays were performed on tumors extracted following drug treatment in patient-derived xenograft (PDX) models of MPNST. Results: Despite a modest effect of SHP2 knockdown on ERK signaling, shPTPN11 reduced MPNST cell growth. SHP2 knockdown or SHP2i treatment alleviated activation of ERK signaling and cyclin D1 expression induced by CDK4/6i, and enhanced the sensitivity to CDK4/6i. Combination benefit was observed in in vitro cell growth and in vivo PDX. Although some PDX models demonstrated similar responses to SHP2i alone or SHP2i + CDK4/6i during the initial four weeks on treatment, we found sustained tumor growth inhibition exerted by the combination on longer therapy. PD studies demonstrated a decrease in p-ERK levels in tumors treated with either SHP2i alone or the SHP2i/CDK4/6i combination, as well as a synergistic suppression of cell cycle regulators by the combination. Conclusions: Our preliminary data demonstrate that the combined inhibition of SHP2 and CDK4/6 is active and produces deep and durable response in models of NF1-associated MPNST. This combination strategy may represent a novel treatment approach for patients with MPNST. Citation Format: Jiawan Wang, Ana Calizo, Kai Pollard, Lindy Zhang, John M. Gross, Nicolas Llosa, Angela C. Hirbe, Christine A. Pratilas. Combined inhibition of SHP2 and CDK4/6 is active in preclinical models of NF1-associated malignant peripheral nerve sheath tumor [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A020.

Abstract IA010: Novel targeted therapy approaches in NF1-MPNST

Abstract NF1 is an essential negative regulator of RAS activity and its function is lost in nearly 90% of malignant peripheral nerve sheath tumors (MPNST). Additional recurrent molecular changes include loss of function alterations in CDKN2A, TP53, EED and SUZ12, but molecular targeting of these genomic events represents a unique challenge. We previously reported that the efficacy of MEK inhibitor is limited by adaptive activation of receptor tyrosine kinases and the adaptor protein SHP2, and that combined inhibition of MEK and SHP2 is effective in MPNST. However, the clinical potential of combination MEK and SHP2 inhibitors may be limited by the overlapping toxicity induced by ERK pathway inhibition. Genetic depletion of PTPN11 has substantial growth inhibitory effects in preclinical models of MPNST, suggesting that SHP2 is an exciting new focus for targeted therapy development in NF1-MPSNT. Loss of CDKN2A, inactivation of RB1, and hyperactivation of cyclin dependent kinases (CDK) in MPNST also suggest that small-molecule CDK4/6 inhibitors (CDK4/6i) may offer a potential combination strategy. Our preclinical data suggest that this combination is active, well-tolerated, and has the potential for immediate advancement into clinical trials. Citation Format: Christine A. Pratilas. Novel targeted therapy approaches in NF1-MPNST [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA010.

Abstract A013: CDK2 and CDK4/6 inhibition in GIST: Mechanisms of response and resistance

Abstract Advanced GIST is characterized by genomic perturbations of key cell cycle regulators. Oncogenic activation of CDK4/6 results in RB1 inactivation and cell cycle progression. Given that single-agent CDK4/6 inhibitor (CDK4/6i) therapy failed to show clinical activity in advanced GIST, we evaluated strategies for maximizing response to therapeutic CDK4/6 inhibition. Targeted next-generation sequencing and multiplexed protein imaging were used to detect cell cycle regulator aberrations in GIST clinical samples (N=18), including 8 metastatic TKI-resistant GISTs. Multiple metastases were analyzed in 3 patients. The impact of CDK2i (CDK2 inhibitor-II), CDK4/6i (palbociclib or abemaciclib), and CDK2/4/6i (PF-06873600) was determined through cell proliferation and protein detection assays in vitro and in vivo. Mechanisms of acquired CDK2i and CDK4/6i resistance were characterized in GIST cell lines after long-term exposure. The results demonstrate recurrent genomic aberrations in cell cycle regulators causing co-activation of the CDK2 and CDK4/6 pathways. Identical aberrations of p16, RB1, and TP53 were present in all metastases from 3 patients. We show that therapeutic co-targeting of CDK2 and CDK4/6 is synergistic in GIST cell lines with intact RB1, through inhibition of RB1 hyperphosphorylation and cell proliferation (P<0.01). Intact RB1 predicted response to treatment, whereas RB1-deficient models were resistant. Moreover, we identify RB1 inactivation and a novel oncogenic cyclin D1 resulting from an intragenic rearrangement (CCND1::chr11.g:70025223) as mechanisms of acquired CDK inhibitor resistance in GIST. The CCND1 rearrangement deleted the cyclin D1 C-terminal Thr286 and Thr288 residues which mediate cyclin D1 proteasomal degradation, resulting in overexpression of an abnormal cyclin D1. CDK inhibitor resistance properties were corroborated by lentiviral transduction of the CCND1 fusion gene into fusion-negative GIST, leiomyosarcoma, and breast cancer cells. These studies establish the biologic rationale for CDK2 and CDK4/6 co-inhibition as therapeutic strategy in patients with advanced GIST, including patients with metastatic GIST progressing on TKIs. In addition, these findings expand the spectrum of potential CDK inhibitor resistance mechanisms with translational potential for improving cell cycle targeted therapies in other cancer types. Citation Format: Inga-Marie Schaefer, Matthew L. Hemming, Meijun Z. Lundberg, Matthew P. Serrata, Isabel Goldaracena, Ninning Liu, Peng Yin, Joao A. Paulo, Steven P. Gygi, Suzanne George, Jeffrey A. Morgan, Monica M. Bertagnolli, Ewa T. Sicinska, Adrian Mariño-Enríquez, Jason L. Hornick, Chandrajit P. Raut, George D. Demetri, Wen-Bin Ou, Sinem K. Saka, Jonathan A. Fletcher. CDK2 and CDK4/6 inhibition in GIST: Mechanisms of response and resistance [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A013.

P53 positively regulates the proliferation of hepatic progenitor cells promoted by laminin-521

Hepatic progenitor cells (HPCs) hold tremendous potential for liver regeneration, but their well-known limitation of proliferation hampers their broader use. There is evidence that laminin is required for the proliferation of HPCs, but the laminin isoform that plays the dominant role and the key intracellular downstream targets that mediate the regulation of HPC proliferation have yet to be determined. Here we showed that p53 expression increased gradually and reached maximal levels around 8 days when laminin α4, α5, β2, β1, and γ1 subunit levels also reached a maximum during HPC activation and expansion. Laminin-521 (LN-521) promoted greater proliferation of HPCs than do laminin, matrigel or other laminin isoforms. Inactivation of p53 by PFT-α or Ad-p53V143A inhibited the promotion of proliferation by LN-521. Further complementary MRI and bioluminescence imaging analysis showed that p53 inactivation decreased the proliferation of transplanted HPCs in vivo. p53 was activated by LN-521 through the Integrin α6β1/FAK-Src-Paxillin/Akt axis. Activated p53 was involved in the nuclear translocation of CDK4 and inactivation of Rb by inducing p27Kip1. Taken together, this study identifies LN-521 as an ideal candidate substrate for HPC culture and uncovers an unexpected positive role for p53 in regulating proliferation of HPCs, which makes it a potential target for HPC-based regenerative medicine.

Emerging Biomarkers and the Changing Landscape of Small Cell Lung Cancer.

Simple SummarySmall cell lung cancer (SCLC) is an aggressive cancer representing 15% of all lung cancers. Unlike other types of lung cancer, treatments for SCLC have changed very little in the past 20 years and therefore, the survival rate remains low. This is due, in part, to the lack of understanding of the biological basis of this disease and the previous idea that all SCLCs are the same. Multiple recent studies have identified that SCLCs have varying biological activity and can be divided into four different groups. The advantage of this is that each of these four groups responds differently to new treatments, which hopefully will dramatically improve survival. Additionally, the aim of these new treatments is to specifically target these biological differences in SCLC so normal/non cancer cells are unaffected, leading to decreased side effects and a better quality of life. There is still a lot unknown about SCLC, but these new findings offer a glimmer of hope for patients in the future.Small cell lung cancer (SCLC) is a high-grade neuroendocrine malignancy with an aggressive behavior and dismal prognosis. 5-year overall survival remains a disappointing 7%. Genomically, SCLCs are homogeneous compared to non-small cell lung cancers and are characterized almost always by functional inactivation of RB1 and TP53 with no actionable mutations. Additionally, SCLCs histologically appear uniform. Thus, SCLCs are currently managed as a single disease with platinum-based chemotherapy remaining the cornerstone of treatment. Recent studies have identified expression of dominant transcriptional signatures which may permit classification of SCLCs into four biologically distinct subtypes, namely, SCLC-A, SCLC-N, SCLC-P, and SCLC-I. These groups are readily detectable by immunohistochemistry and also have potential predictive utility for emerging therapies, including PARPi, immune checkpoint inhibitors, and DLL3 targeted therapies. In contrast with their histology, studies have identified that SCLCs display both inter- and intra-tumoral heterogeneity. Identification of subpopulations of cells with high expression of PLCG2 has been linked with risk of metastasis. SCLCs also display subtype switching under therapy pressure which may contribute furthermore to metastatic ability and chemoresistance. In this review, we summarize the recent developments in the understanding of the biology of SCLCs, and discuss the potential diagnostic, prognostic, and treatment opportunities the four proposed subtypes may present for the future. We also discuss the emerging evidence of tumor heterogeneity and plasticity in SCLCs which have been implicated in metastasis and acquired therapeutic resistance seen in these aggressive tumors.

Hepatocellular carcinoma evades RB1-induced senescence by activating the FOXM1-FOXO1 axis.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers. The retinoblastoma protein (RB1), a regulator of cell proliferation, is functionally inactivated in HCC by CYCLIN D/E-mediated phosphorylation. However, the mechanism of RB1-inactivation is unclear because only small percentages of HCCs exhibit amplification of CYCLIN D/E or mutations in the CDK-inhibitory genes. We show that FOXM1, which is overexpressed and critical for HCC, plays essential roles in inactivating RB1 and suppressing RB1-induced senescence of the HCC cells. Mechanistically, FOXM1 binds RB1 and DNMT3B to repress the expression of FOXO1, leading to a decrease in the levels of the CDK-inhibitors, creating an environment for phosphorylation and inactivation of RB1. Consistent with that, inhibition of FOXM1 causes increased expression of FOXO1 with consequent activation of RB1, leading to senescence of the HCC cells, in vitro and in vivo. Also, repression-deficient mutants of FOXM1 induce senescence that is blocked by depletion of RB1 or FOXO1. We provide evidence that human HCCs rely upon this FOXM1-FOXO1 axis for phosphorylation and inactivation of RB1. The observations demonstrate the existence of a new autoregulatory loop of RB1-inactivation in HCC involving a FOXM1-FOXO1 axis that is required for phosphorylation of RB1 and for aggressive progression of HCC.

Genomic landscape of non-small-cell lung cancer with methylthioadenosine phosphorylase (MTAP) deficiency.

New treatment strategies for advanced non-small-cell lung carcinoma (NSCLC) include synthetic lethality targets focused on protein arginine methyl transferases such as PRMT5 that exploit the impact of genomic loss of methylthioadenosine phosphorylase (MTAP). Twenty nine thousand three hundred seventy nine advanced NSCLC cases underwent hybrid-capture based comprehensive genomic profiling between June 1, 2018 and May 31, 2020. PD-L1 expression was determined by immunohistochemistry (Dako 22C3 PharmDx assay). 13.4% (3928/29,379) NSCLC cases exhibited MTAP loss distributed in adenocarcinoma (59%), squamous cell carcinoma (22%), NSCLC not otherwise specified (16%), and 1% each for large-cell neuroendocrine, sarcomatoid, and adenosquamous carcinoma. Statistically significant differences in mitogenic driver alterations included more KRAS G12C mutations in MTAP-intact versus MTAP-lost (12% vs. 10%, p=0.0003) and fewer EGFR short variant mutations in MTAP-intact versus MTAP-lost NSCLC (10% vs. 13%, p < 0.0001). Statistically significant differences in currently untargetable genomic alterations included higher frequencies of TP53 (70% vs. 63%, p < 0.0001) and RB1 inactivation (10% vs. 2%, p < 0.0001) in MTAP-intact compared to MTAP-lost NSCLC. SMARCA4 inactivation (7% vs. 10%, p < 0.0001) was less frequent in MTAP-intact versus MTAP-lost NSCLC. Alterations in ERBB2, MET, ALK, ROS1, and NTRK1 did not significantly differ between the two groups. Predictors of immunotherapy efficacy were higher in MTAP-intact versus MTAP-lost NSCLC including tumor mutational burden (9.4 vs. 8.6 mut/Mb, p=0.001) and low (30% vs. 28%, p=0.01) and high PD-L1 (32% vs. 30%, p=0.01) expression. Alterations in biomarkers potentially predictive of immune checkpoint inhibitor resistance (STK11, KEAP1, and MDM2) were similar in the two groups. MTAP loss occurs in 13% of NSCLC, supporting the development of targeted therapies to exploit PRMT5 hyper-dependence. MTAP loss is accompanied by small differences in targeted and immunotherapy options which may impact future combination strategies.

Whole exome analysis reveals the genomic profiling related to chemo-resistance in Chinese population with limited-disease small cell lung cancer.

The mechanism of chemo-resistance in small cell lung cancer (SCLC) is unclear. This study aims to explore the resistance-related genomic profiles of residual tumors after neo-adjuvant chemotherapy (NAC) in SCLC through the whole-exome sequencing (WES). A total of 416 limited diseases (LD) SCLC patients underwent surgery were retrospectively analyzed, of which 40 patients received NAC. Then we selected 29 patients undergoing NAC (n=19) and chemotherapy naïve (CTN, n=10) to perform WES sequence with formalin-fixed paraffin-embedded samples including tumor and paired para-tumor. In total, single nucleotide variation and mutation rate were similar between NAC and CTN groups. The mutation signatures were significantly discrepant between NAC and CTN groups, as well as among patients with partial response (PR), stable disease (SD), and progressive disease. There were more copy number variation deletions in NAC group compared with CTN group. The inactivation of TP53 and RB1 were the most significantly events in both NAC and CTN groups. RB1 nonsense mutations were recurrent in NAC group (9/19 vs. 0/9, 47.4% vs. 0%) with favorable survival, while the frame-shift deletions were frequent in CTN group (3/9 vs. 3/19, 33.3% vs.15.8%). Integrated function enrichment revealed that the frequently mutant genes were involved in cell cycle, metabolic reprogramming, and oncogenic signaling pathways in NAC group, such as BTG2 pathway, glycolysis in senescence and P53 pathway. A total of 27 genes presented frequently mutant in NAC group and might played a positive role in drug resistance. Multiple genes including BRINP3, MYH6, ST18, and PCHD15, which were associated with prognosis, occurred mutant frequently in PR and SD groups. Residual tumors after neo-adjuvant therapy exhibited different mutation signature spectrum. Multiple genes including RB1 nonsense mutations, BRINP3, MYH6, ST18, and PCHD15 were with frequent mutation in residual tumors, which might participate chemo-resistance and influenced the prognosis in patients with limited disease SCLC.

Abstract 5648: Response and resistance to CDK2 and CDK4/6 inhibition in GIST

Abstract Gastrointestinal stromal tumor (GIST) is the most common GI sarcoma and is generally initiated by KIT or PDGFRA mutations which are compelling therapeutic targets for tyrosine kinase inhibitors (TKI). However, the emergence of secondary mutations results in clinical resistance to available TKIs. GIST progression is driven by genomic events which incrementally target the p16-CDK4/6-RB1 and p14-TP53-RB1 pathways to create CDK4/6 and CDK2 oncogenic co-dependency. Based on limited efficacy of single-agent CDK4/6-inhibitor (CDK4/6i) therapy in GIST, we evaluated strategies of co-targeting CDK2 and CDK4/6. Multiplexed protein imaging (via Immuno-SABER) was validated for the detection of cell cycle regulator aberrations in GIST clinical samples (N=18), 7 of which were TKI-resistant, and including 3 patients in whom multiple metastases were analyzed. The impact of various CDK perturbants using CDK2i (CDK2 inhibitor-II), CDK4/6i (palbociclib or abemaciclib), and CDK2/4/6i (PF-06873600) was determined through cell proliferation and protein detection assays in GIST cell lines and murine xenografts. Mechanisms of acquired CDK2i and CDK4/6i resistance were characterized in GIST cell lines after long-term exposure. Abnormal expression/biallelic inactivation of CDKN2A/p16, RB1, and TP53 were identified in 7 (39%), 2 (11%), and 2 (11%) of 18 GISTs, respectively. Identical aberrations of p16, RB1, and TP53 were present in all metastases from 3 patients. Since 5 of 7 RB1-intact advanced GISTs had co-dysregulation of the CDK2 and CDK4/6 pathways, we evaluated co-inhibition of CDK2 and CDK4/6 in vitro and in vivo which inhibited cell proliferation (P&amp;lt;0.01) and RB1 hyperphosphorylation. Intact RB1 predicted response to treatment, whereas RB1-deficient models were resistant. Two resistant sub-lines emerged after 11 and 14 months of palbociclib exposure: one with biallelic genomic RB1 inactivation and the other with the first known example of a cyclin D1 coding sequence fusion with oncogenic properties (CCND1::chr11.g:70025223). The CCND1 fusion deleted the cyclin D1 C-terminal Thr286 and Thr288 residues which mediate cyclin D1 proteasomal degradation, resulting in overexpression of an abnormal cyclin D1. Palbociclib-resistance properties were corroborated by lentiviral transduction of the CCND1 fusion gene into fusion-negative GIST, leiomyosarcoma, and breast cancer cells. CDK2 and CDK4/6 pathway perturbations with retained RB1 are frequent in advanced GIST and can be conserved across metastases, creating a compelling biologic rationale for therapeutic cell cycle restoration. We show that co-inhibition of CDK2 and CDK4/6 is synergistic in GIST and highlight RB1 inactivation and a novel oncogenic cyclin D1 as mechanisms of acquired CDKi resistance. Hence, combination therapies targeting CDK2 and CDK4/6 with correlative biomarkers predictive of response should be evaluated in patients with metastatic or TKI-resistant GIST. Citation Format: Inga-Marie Schaefer, Meijun Z. Lundberg, Matthew L. Hemming, Sinem K. Saka, Matthew P. Serrata, Isabel Goldaracena, Ninning Liu, Peng Yin, Joao A. Paulo, Steven Gygi, George D. Demetri, Ewa Sicinska, Adrian Mariño-Enríquez, Jason L. Hornick, Chandrajit P. Raut, Wen-Bin Ou, Jonathan A. Fletcher. Response and resistance to CDK2 and CDK4/6 inhibition in GIST [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 5648.

Abstract 41: Multi-focal genomic dissection of synchronous primary and metastatic tissue from de novo metastatic prostate cancer

Abstract Background: 10% of newly diagnosed prostate cancer presents with metastases. Known as de novo metastatic castrate-sensitive prostate cancer (mCSPC), it is disproportionally responsible for &amp;gt;50% of prostate cancer deaths. Cancer genotyping can identify vulnerabilities exploitable by targeted therapies, and promises to help prognosticate. However, tissue from de novo mCSPC is scarce; neither prostatectomy nor metastatic biopsy is standard, and it is unknown if diagnostic biopsies are representative of synchronous metastases. The potential for plasma circulating tumor DNA (ctDNA) to inform on tumor genotype is also unknown. Methods: We performed comprehensive pathological and genomic assessment of all spatially or phenotypically-distinct tumor foci (n=523) in 43 patients with de novo mCSPC who underwent prostatectomy, pelvic lymph node dissection, and plasma collection. Results: 91% (478/523) of tissue foci had evidence of prostate cancer by targeted DNA sequencing, with a median tumor fraction of 48%. When modeling random selection of a single primary foci (mirroring biopsy tissue availability in clinic), tumor fraction was &amp;lt;20% in 19% of patients. Only 46% of plasma cell-free DNA samples prior to systemic therapy had a ctDNA fraction above 0.3% (limit of detection); median tumor fraction of 5% in samples with confirmed ctDNA. We observed recurrent alterations in major driver genes, including TP53, FOXA1, PTEN, and RB1, and the genomic landscape was very similar to published cohorts of castration-resistant prostate cancer (excluding AR). Primary site genomic heterogeneity was pervasive, including secondary (clonally distinct) prostate cancer populations in 14% of patients. Polyclonal seeding of metastases was detected in 26% of patients. Biallelic inactivation of TP53, PTEN, and/or RB1 was observed in 63% of tumors, and was frequently found in synchronous metastases and ctDNA. The two patients with compound disruption of TP53, PTEN, and RB1 experienced rapid progression to castration-resistance and death within two years of diagnosis, despite initial low-risk clinical features. Across the cohort, biallelic disruption of TP53 together with high-risk clinical features at diagnosis was associated with rapid progression (HR 4.64 (95% CI: 1.70-12.69); P = 0.003). Conclusions: One fifth of patients with de novo mCSPC have pervasive low tumor fraction in their primary tumor and blood plasma. Many tumors exhibit spatial heterogeneity within the primary site, with evidence of multiple clones seeding metastases. This data raises concerns about accurate tumor genotyping in routine clinical practice where needle biopsy specimens are the only available tissue for profiling. Nevertheless, some de novo mCSPC are marked by aggressive genomics and experience rapid progression to lethal disease, suggesting that tailored multi-focal genomic profiling can further segment the disease. Citation Format: Evan Warner, Kim Van der Eecken, Andrew J. Murtha, Edmond M. Kwan, Sarah W. Ng, Xinyi E. Chen, Cecily Q. Bernales, Grainne Donnellan, Elena Schonlau, Sofie Verbeke, Nicolaas Lumen, Jo Van Dorpe, Gillian Vandekerkhove, Elie Ritch, Matti Annala, Bram De Laere, Piet Ost, Alexander W. Wyatt. Multi-focal genomic dissection of synchronous primary and metastatic tissue from de novo metastatic prostate cancer [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 41.

Abstract 1672: Effect of BCOR loss in Xenopus laevis and tropicalis: Insights for retinal development and retinoblastoma

Abstract Retinoblastoma is the most common pediatric eye cancer, accounting for approximately 11% of cancers within the first year of life. Almost all retinoblastoma tumors are initiated by bi-allelic mutational inactivation of RB1, yet there is a broad range of clinical presentations, suggesting the existence of secondary driver events in cancer progression. Genomic analysis identified BCOR as the second most commonly mutated gene in retinoblastoma. There is growing evidence that inactivating BCOR mutations are associated with more aggressive features, such as dedifferentiation, vitreous seeding, optic nerve invasion, and worse patient outcomes. Unfortunately, little is known about the function of BCOR in the retina and the effects of BCOR loss in retinoblastoma. Here, we used Xenopus laevis and tropicalis models to study the role of BCOR in normal ocular and retinal development. In both models, targeted depletion of BCOR in cell lineages giving rise to the eye resulted in a discrete microphthalmia phenotype. Furthermore, we developed in situ hybridization tools for probing characteristic retinal lineage-specific genes and performed histology to compare structural differences. We provide evidence that BCOR normally regulates critical steps in retinal developmental, suggesting that loss of BCOR in retinoblastoma may drive tumor progression by disrupting this function. Citation Format: Michelle Garying Zhang, Dawn Owens, Nicole Jones, Daniel Pelaez, J. William Harbour. Effect of BCOR loss in Xenopus laevis and tropicalis: Insights for retinal development and retinoblastoma [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 1672.

Abstract 2557: Slit2 inhibits small cell lung cancer by targeting tumor microenvironment

Abstract Small cell lung cancer (SCLC) is the deadliest subtype of lung cancer with a 5-year survival rate of &amp;lt;5% in extensive-state disease and is designated by US Congress as a “recalcitrant cancer”. Recently approved by FDA, immune checkpoint blockade (ICB) immunotherapy showed marginally improved survival and only in a few patients, suggesting an inert immune tumor microenvironment (TME) and the existence of SCLC subtypes. Therefore, there is a significant need to develop more effective therapies for SCLC. In addition to the nearly universal biallelic inactivation of Rb and Tp53 genes in human SCLC tumors, comprehensive genome-wide screening studies have identified major co-occurring mutations in PTEN, CREBBP, EP300, SLIT2, MLL, COBL, and EPHA7 genes. Among them, SLIT2 showed the highest level of genomic abnormalities defined by a pronounced clustering of mutations and frequent genomic loss. However, the role of Slit2 in SCLC has not been defined yet. By using genetically engineered human SCLC cells overexpressing Slit2 or recombinant Slit2 (rSlit2) protein, we show that Slit2 inhibits SCLC cells proliferation, migration, and invasion properties and tumor sphere formation ability of drug-resistant SCLC cells in vitro. The mechanistic studies have identified inhibition of AKT and β-catenin mediated signaling pathways by Slit2 overexpression. Next, we implanted human SCLC cell lines into the immunocompromised NSG mice subcutaneously followed by treatment with rSlit2 or sterile saline. Analysis of tumor volume over the 4 weeks showed that rSlit2 treatment significantly reduced the growth of SCLC tumors. Further analysis of tumors showed that rSlit2 treated tumors harbor less M2-like CD206+ macrophages. Overall, these results suggest that Slit2 possesses tumor suppressive properties and could be developed as a novel therapeutic agent against SCLC. Citation Format: Dinesh K. Ahirwar, Manish Charan, Swati Misri, Konstantin Shilo, Ramesh Ganju. Slit2 inhibits small cell lung cancer by targeting tumor microenvironment [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 2557.

Insights into mutational differences between upper tract urothelial carcinoma and urothelial carcinoma of the bladder.

e15125 Background: Upper tract urothelial carcinoma (UTUC) is histologically similar to bladder urothelial carcinoma (BUC), yet several clinical, biological and molecular features are unique to UTUC. UTUC may have unique genomic factors compared to BUC, while major knowledge gaps remain in our understanding of the biology and genomic landscape of UTUC. Methods: We retrospectively sequenced tumor samples and matched germline DNA using targeted next-generation sequencing methods and analyzed at least 642 cancer-associated genes. The cohort included 35 UTUC patients and 36 BUC patients. Results: Among all cases, KMT2D and TP53 were the most common mutations (account for 66% and 75% in UTUC and BUC, respectively). The most frequently mutated genes in UTUC were KMT2D (37%), TP53 (29%), ELF3 (26%), FGFR3 (23%), ASXL1 (20%), DKN2A (20%), DKN2B (17%), REBBP (17%), MT3B (17%), GNAS (17%) and PIK3CA (17%). The most frequently mutated genes in BUC were KMT2D (36%), TP53 (39%), MUC17 (14%), ARID1A (22%), GNAS (19%), RB1 (19%), BCL6 (19%), E2F3 (19%), EIF4A2 (19%) and FGF3 (19%). However significant differences in the prevalence of mutations in individual genes were observed. FGFR3 (23% vs. 8%) and ELF3 (26% vs. 11%) were more frequently altered, whereas RB1 (6% vs. 19%), E2F3 (3% vs. 19%) and BCL6 (3% vs. 19%) were less frequently altered in UTUC (P &lt; 0.05). FGFR3 mutations have been shown to be oncogenic and RB1 inactivation has been associated with genomic instability in urothelial cancer in previous researches, which represent potential therapeutic targets. Conclusions: Â UTUC and UCB exhibit significant differences in the prevalence of genomic landscape and carcinogenesis. A comprehensive understanding of the biology of UTUC and UCB is needed to identify new drug targets in order to improve clinical outcomes.

Comparative characteristics of small cell lung cancer and Ewing's sarcoma: a narrative review.

Background and ObjectiveSmall cell lung cancer (SCLC) and Ewing’s sarcoma (ES) at the disseminated stage are not amenable to therapy and have a dismal prognosis with low survival rates. Despite representing different tumor entities, treatment for both malignancies relies on cytotoxic chemotherapy that has not considerably changed for the past decades. The genomic background has been extensively studied and found to comprise inactivation of p53 and RB1 in case of SCLC and EWSR1/FLI1 rearrangement in case of ES resulting in aggressive tumors in adults with heavy tobacco consumption and as bone tumor in juveniles, respectively. New therapeutic modalities are urgently needed to improve the outcomes of both tumor entities, especially in patients with metastatic disease or recurrences. This review summarizes the common cell biologic and clinical characteristics of difficult-to-treat SCLC and ES and discusses their refractoriness and options to improve the therapeutic efficacy.MethodsPubMed and Euro PMC were searched from January 1st, 2012 to January 16th, 2022 using the following key words: “SCLC”, “Ewing´s sarcoma”, “Genomics” and “Chemoresistance” as well as own work.Key Content and FindingsTherapy of SCLC and ES involves the use of undirected cytotoxic drugs in multimodal chemotherapy and administration of topotecan for 2nd line SCLC regimens. Despite highly aggressive chemotherapies, outcomes are dismal for patients with disseminated tumors. A host of unrelated drugs and targeted therapeutics have failed to result in progress for the patients and the underlying mechanisms of chemoresistance are still not clear. Identification of chemoresistance-reversing modulators in vitro and patient-derived xenografts of SCLC and ES has not translated into new therapies.ConclusionsThe global chemoresistance of SCLC and ES may be explained by physiological resistance at the tumor level and formation of larger spheroids that contain quiescent and hypoxic tumor cells in regions that occlude therapeutics. This type of chemoresistance is difficult to overcome and prevent the accumulation of effective drug concentration at the tumor cell level to a significant degree leaving therapeutic interventions of any kind ineffective.

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.

RB1-Negative Retinal Organoids Display Proliferation of Cone Photoreceptors and Loss of Retinal Differentiation.

Simple SummaryRetinoblastoma is a tumor of the eye’s retina, which is the very specialized tissue responsible for vision. In 98% of cases, the tumor is caused by inactivation of the RB1 gene. Due to lack of material and models, the understanding of RB1 mutations in tumor development is still unsatisfactory. We aimed to establish a human laboratory model for retinoblastoma. While differentiating stem cells with a mutation in RB1 into retina, we observed reduced differentiation potential but enhanced proliferation—general hallmarks of tumor development. The gene expression signature in the model resembled that of tumor material. This approach now enables research on retinoblastoma and probably therapy in the correct tissue, the human retina.Retinoblastoma is a tumor of the eye in children under the age of five caused by biallelic inactivation of the RB1 tumor suppressor gene in maturing retinal cells. Cancer models are essential for understanding tumor development and in preclinical research. Because of the complex organization of the human retina, such models were challenging to develop for retinoblastoma. Here, we present an organoid model based on differentiation of human embryonic stem cells into neural retina after inactivation of RB1 by CRISPR/Cas9 mutagenesis. Wildtype and RB1 heterozygous mutant retinal organoids were indistinguishable with respect to morphology, temporal development of retinal cell types and global mRNA expression. However, loss of pRB resulted in spatially disorganized organoids and aberrant differentiation, indicated by disintegration of organoids beyond day 130 of differentiation and depletion of most retinal cell types. Only cone photoreceptors were abundant and continued to proliferate, supporting these as candidate cells-of-origin for retinoblastoma. Transcriptome analysis of RB1 knockout organoids and primary retinoblastoma revealed gain of a retinoblastoma expression signature in the organoids, characterized by upregulation of RBL1 (p107), MDM2, DEK, SYK and HELLS. In addition, genes related to immune response and extracellular matrix were specifically upregulated in RB1-negative organoids. In vitro retinal organoids therefore display some features associated with retinoblastoma and, so far, represent the only valid human cancer model for the development of this disease.

Retinoblastoma patient-derived stem cells-an in vivo model to study the role of RB1 in adipogenesis.

Retinoblastoma (RB1) protein is a multifunctional protein that plays an important role in cell cycle regulation and cell differentiation, including adipogenesis. A detailed literature search to understand the role of RB1 in adipogenesis revealed that the nature of the RB1 inactivation (invivo/invitro) led to differences in adipogenesis. The majority of these studies were animal-based, and the only study in humans employed an invitro mode of RB1 inactivation. To overcome these differences and lack of human studies, we sought to explore the role of RB1 in adipogenesis using orbital adipose mesenchymal stem cells (OAMSCs) from patients with retinoblastoma that innately carry a heterozygous RB1 mutation. We hypothesized that these patient-derived RB1 mutant OAMSCs can model invivo RB1 inactivation in humans. Our study revealed increased adipogenesis with a bias toward brown adipogenesis in the RB1 mutant in addition to an increased number of adipocytes in the mitotic phase.