Lead compound profiling for small molecule inhibitors of the REV1-CT/RIR Translesion synthesis Protein-Protein interaction.

Translesion synthesis (TLS) is a cellular mechanism through which actively replicating cells recruit specialized, low-fidelity DNA polymerases to damaged DNA to allow for replication past these lesions. REV1 is one of these TLS DNA polymerases that functions primarily as a scaffolding protein to organize the TLS heteroprotein complex and ensure replication occurs in the presence of DNA lesions. The C-Terminal domain of REV1 (REV1-CT) forms many protein-protein interactions (PPIs) with other TLS polymerases, making it essential for TLS function and a promising drug target for anti-cancer drug development. We utilized several lead identification strategies to identify various small molecules capable of disrupting the PPI between REV1-CT and the REV1 Interacting Regions (RIR) present in several other TLS polymerases. These lead compounds were profiled in several in vitro potency and PK assays to identify two scaffolds (1 and 6) as the most promising for further development. Both 1 and 6 synergized with cisplatin in a REV1-dependent fashion and demonstrated promising in vivo PK and toxicity profiles.

Acquired Cross-Resistance in Small Cell Lung Cancer due to Extrachromosomal DNA Amplification of MYC Paralogs.

Small cell lung cancer (SCLC) presents as a highly chemosensitive malignancy but acquires cross-resistance after relapse. This transformation is nearly inevitable in patients but has been difficult to capture in laboratory models. Here, we present a preclinical system that recapitulates acquired cross-resistance, developed from 51 patient-derived xenograft (PDX) models. Each model was tested in vivo against three clinical regimens: cisplatin plus etoposide, olaparib plus temozolomide, and topotecan. These drug-response profiles captured hallmark clinical features of SCLC, such as the emergence of treatment-refractory disease after early relapse. For one patient, serial PDX models revealed that cross-resistance was acquired through MYC amplification on extrachromosomal DNA (ecDNA). Genomic and transcriptional profiles of the full PDX panel revealed that MYC paralog amplifications on ecDNAs were recurrent in relapsed cross-resistant SCLC, and this was corroborated in tumor biopsies from relapsed patients. We conclude that ecDNAs with MYC paralogs are recurrent drivers of cross-resistance in SCLC. SIGNIFICANCE: SCLC is initially chemosensitive, but acquired cross-resistance renders this disease refractory to further treatment and ultimately fatal. The genomic drivers of this transformation are unknown. We use a population of PDX models to discover that amplifications of MYC paralogs on ecDNA are recurrent drivers of acquired cross-resistance in SCLC. This article is featured in Selected Articles from This Issue, p. 695.

Patterns in the tapestry of chromatin-bound RB.

The retinoblastoma protein (RB)-mediated regulation of E2F is a component of a highly conserved cell cycle machine. However, RB's tumor suppressor activity, like RB's requirement in animal development, is tissue-specific, context-specific, and sometimes appears uncoupled from cell proliferation. Detailed new information about RB's genomic distribution provides a new perspective on the complexity of RB function, suggesting that some of its functional specificity results from context-specific RB association with chromatin. Here we summarize recent evidence showing that RB targets different types of chromatin regulatory elements at different cell cycle stages. RB controls traditional RB/E2F targets prior to S-phase, but, when cells proliferate, RB redistributes to cell type-specific chromatin loci. We discuss the broad implications of the new data for RB research.

Acquired Cross-resistance in Small Cell Lung Cancer due to Extrachromosomal DNA Amplification of MYC paralogs.

Small cell lung cancer (SCLC) presents as a highly chemosensitive malignancy but acquires cross-resistance after relapse. This transformation is nearly inevitable in patients but has been difficult to capture in laboratory models. Here we present a pre-clinical system that recapitulates acquired cross-resistance in SCLC, developed from 51 patient-derived xenografts (PDXs). Each model was tested for in vivo sensitivity to three clinical regimens: cisplatin plus etoposide, olaparib plus temozolomide, and topotecan. These functional profiles captured hallmark clinical features, such as the emergence of treatment-refractory disease after early relapse. Serially derived PDX models from the same patient revealed that cross-resistance was acquired through a MYC amplification on extrachromosomal DNA (ecDNA). Genomic and transcriptional profiles of the full PDX panel revealed that this was not unique to one patient, as MYC paralog amplifications on ecDNAs were recurrent among cross-resistant models derived from patients after relapse. We conclude that ecDNAs with MYC paralogs are recurrent drivers of cross-resistance in SCLC. SIGNIFICANCE: SCLC is initially chemosensitive, but acquired cross-resistance renders this disease refractory to further treatment and ultimately fatal. The genomic drivers of this transformation are unknown. We use a population of PDX models to discover that amplifications of MYC paralogs on ecDNA are recurrent drivers of acquired cross-resistance in SCLC.

Therapy-induced APOBEC3A drives evolution of persistent cancer cells.

Acquired drug resistance to anticancer targeted therapies remains an unsolved clinical problem. Although many drivers of acquired drug resistance have been identified1-4, the underlying molecular mechanisms shaping tumour evolution during treatment are incompletely understood. Genomic profiling of patient tumours has implicated apolipoprotein B messenger RNA editing catalytic polypeptide-like (APOBEC) cytidine deaminases in tumour evolution; however, their role during therapy and the development of acquired drug resistance is undefined. Here we report that lung cancer targeted therapies commonly used in the clinic can induce cytidine deaminase APOBEC3A (A3A), leading to sustained mutagenesis in drug-tolerant cancer cells persisting during therapy. Therapy-induced A3A promotes the formation of double-strand DNA breaks, increasing genomic instability in drug-tolerant persisters. Deletion of A3A reduces APOBEC mutations and structural variations in persister cells and delays the development of drug resistance. APOBEC mutational signatures are enriched in tumours from patients with lung cancer who progressed after extended responses to targeted therapies. This study shows that induction of A3A in response to targeted therapies drives evolution of drug-tolerant persister cells, suggesting that suppression of A3A expression or activity may represent a potential therapeutic strategy in the prevention or delay of acquired resistance to lung cancer targeted therapy.

Seeing is believing: the impact of RB on nuclear organization.

The retinoblastoma tumor suppressor (RB) prevents G1 to S cell cycle transition by inhibiting E2F activity. This function requires that RB remains un- or underphosphorylated (the so-called active forms of RB). Recently, we showed that active forms of RB cause widespread changes in nuclear architecture that are visible under a microscope. These phenotypes did not correlate with cell cycle arrest or repression of the E2F transcriptional program, but appeared later, and were associated with the appearance of autophagy or in IMR-90 cells with senescence markers. In this perspective, we describe the relative timing of these RB-induced events and discuss the mechanisms that may underlie RB-induced chromatin dispersion. We consider the relationship between RB-induced dispersion, autophagy, and senescence and the potential connection between dispersion and cell cycle exit.

Chromatin-bound protein colocalization analysis using bedGraph2Cluster and PanChIP.

Computational pipelines for chromatin immunoprecipitation sequencing analysis can neglect colocalization events that occur in a mere subset of the genome. Here, we detail a streamlined approach for assessing colocalization of chromatin-bound proteins using the bedGraph2Cluster and PanChIP algorithms. Using histone modifications as an example, bedGraph2Cluster performs clustering analysis on chromatin binding patterns of target proteins. PanChIP then compares these clusters with a reference library of chromatin binding patterns and measures the overlap in peaks, capturing the heterogeneity in chromatin binding and colocalization patterns. For complete details on the use and execution of this protocol, please refer to Sanidas et al. (2022).1.

Chromatin-bound RB targets promoters, enhancers, and CTCF-bound loci and is redistributed by cell-cycle progression.

The interaction of RB with chromatin is key to understanding its molecular functions. Here, for first time, we identify the full spectrum of chromatin-bound RB. Rather than exclusively binding promoters, as is often described, RB targets three fundamentally different types of loci (promoters, enhancers, and insulators), which are largely distinguishable by the mutually exclusive presence of E2F1, c-Jun, and CTCF. While E2F/DP facilitates RB association with promoters, AP-1 recruits RB to enhancers. Although phosphorylation in CDK sites is often portrayed as releasing RB from chromatin, we show that the cell cycle redistributes RB so that it enriches at promoters in G1 and at non-promoter sites in cycling cells. RB-bound promoters include the classic E2F-targets and are similar between lineages, but RB-bound enhancers associate with different categories of genes and vary between cell types. Thus, RB has a well-preserved role controlling E2F in G1, and it targets cell-type-specific enhancers and CTCF sites when cells enter S-phase.

Translesion DNA synthesis mediates acquired resistance to olaparib plus temozolomide in small cell lung cancer.

In small cell lung cancer (SCLC), acquired resistance to DNA-damaging therapy is challenging to study because rebiopsy is rarely performed. We used patient-derived xenograft models, established before therapy and after progression, to dissect acquired resistance to olaparib plus temozolomide (OT), a promising experimental therapy for relapsed SCLC. These pairs of serial models reveal alterations in both cell cycle kinetics and DNA replication and demonstrate both inter- and intratumoral heterogeneity in mechanisms of resistance. In one model pair, up-regulation of translesion DNA synthesis (TLS) enabled tolerance of OT-induced damage during DNA replication. TLS inhibitors restored sensitivity to OT both in vitro and in vivo, and similar synergistic effects were seen in additional SCLC cell lines. This represents the first described mechanism of acquired resistance to DNA damage in a patient with SCLC and highlights the potential of the serial model approach to investigate and overcome resistance to therapy in SCLC.

Active RB causes visible changes in nuclear organization.

RB restricts G1/S progression by inhibiting E2F. Here, we show that sustained expression of active RB, and prolonged G1 arrest, causes visible changes in chromosome architecture that are not directly associated with E2F inhibition. Using FISH probes against two euchromatin RB-associated regions, two heterochromatin domains that lack RB-bound loci, and two whole-chromosome probes, we found that constitutively active RB (ΔCDK-RB) promoted a more diffuse, dispersed, and scattered chromatin organization. These changes were RB dependent, were driven by specific isoforms of monophosphorylated RB, and required known RB-associated activities. ΔCDK-RB altered physical interactions between RB-bound genomic loci, but the RB-induced changes in chromosome architecture were unaffected by dominant-negative DP1. The RB-induced changes appeared to be widespread and influenced chromosome localization within nuclei. Gene expression profiles revealed that the dispersion phenotype was associated with an increased autophagy response. We infer that, after cell cycle arrest, RB acts through noncanonical mechanisms to significantly change nuclear organization, and this reorganization correlates with transitions in cellular state.

E2F/Dp inactivation in fat body cells triggers systemic metabolic changes.

The E2F transcription factors play a critical role in controlling cell fate. In Drosophila, the inactivation of E2F in either muscle or fat body results in lethality, suggesting an essential function for E2F in these tissues. However, the cellular and organismal consequences of inactivating E2F in these tissues are not fully understood. Here, we show that the E2F loss exerts both tissue-intrinsic and systemic effects. The proteomic profiling of E2F-deficient muscle and fat body revealed that E2F regulates carbohydrate metabolism, a conclusion further supported by metabolomic profiling. Intriguingly, animals with E2F-deficient fat body had a lower level of circulating trehalose and reduced storage of fat. Strikingly, a sugar supplement was sufficient to restore both trehalose and fat levels, and subsequently rescued animal lethality. Collectively, our data highlight the unexpected complexity of E2F mutant phenotype, which is a result of combining both tissue-specific and systemic changes that contribute to animal development.

Clinical Outcomes With Abemaciclib After Prior CDK4/6 Inhibitor Progression in Breast Cancer: A Multicenter Experience.

BACKGROUND: Inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6i) are widely used as first-line therapy for hormone receptor-positive metastatic breast cancer (HR+ MBC). Although abemaciclib monotherapy is also FDA-approved for treatment of disease progression on endocrine therapy, there is limited insight into the clinical activity of abemaciclib after progression on prior CDK4/6i. PATIENTS AND METHODS: We identified patients with HR+ MBC from 6 cancer centers in the United States who received abemaciclib after disease progression on prior CDK4/6i, and abstracted clinical features, outcomes, toxicity, and predictive biomarkers. RESULTS: In the multicenter cohort, abemaciclib was well tolerated after a prior course of CDK4/6i (palbociclib)-based therapy; a minority of patients discontinued abemaciclib because of toxicity without progression (9.2%). After progression on palbociclib, most patients (71.3%) received nonsequential therapy with abemaciclib (with ≥1 intervening non-CDK4/6i regimens), with most receiving abemaciclib with an antiestrogen agent (fulvestrant, 47.1%; aromatase inhibitor, 27.6%), and the remainder receiving abemaciclib monotherapy (19.5%). Median progression-free survival for abemaciclib in this population was 5.3 months and median overall survival was 17.2 months, notably similar to results obtained in the MONARCH-1 study of abemaciclib monotherapy in heavily pretreated HR+/HER2-negative CDK4/6i-naïve patients. A total of 36.8% of patients received abemaciclib for ≥6 months. There was no relationship between the duration of clinical benefit while on palbociclib and the subsequent duration of treatment with abemaciclib. RB1, ERBB2, and CCNE1 alterations were noted among patients with rapid progression on abemaciclib. CONCLUSIONS: A subset of patients with HR+ MBC continue to derive clinical benefit from abemaciclib after progression on prior palbociclib. These results highlight the need for future studies to confirm molecular predictors of cross-resistance to CDK4/6i therapy and to better characterize the utility of abemaciclib after disease progression on prior CDK4/6i.

PTEN Loss Mediates Clinical Cross-Resistance to CDK4/6 and PI3Kα Inhibitors in Breast Cancer.

The combination of CDK4/6 inhibitors with antiestrogen therapies significantly improves clinical outcomes in ER-positive advanced breast cancer. To identify mechanisms of acquired resistance, we analyzed serial biopsies and rapid autopsies from patients treated with the combination of the CDK4/6 inhibitor ribociclib with letrozole. This study revealed that some resistant tumors acquired RB loss, whereas other tumors lost PTEN expression at the time of progression. In breast cancer cells, ablation of PTEN, through increased AKT activation, was sufficient to promote resistance to CDK4/6 inhibition in vitro and in vivo. Mechanistically, PTEN loss resulted in exclusion of p27 from the nucleus, leading to increased activation of both CDK4 and CDK2. Because PTEN loss also causes resistance to PI3Kα inhibitors, currently approved in the post-CDK4/6 setting, these findings provide critical insight into how this single genetic event may cause clinical cross-resistance to multiple targeted therapies in the same patient, with implications for optimal treatment-sequencing strategies. SIGNIFICANCE: Our analysis of serial biopsies uncovered RB and PTEN loss as mechanisms of acquired resistance to CDK4/6 inhibitors, utilized as first-line treatment for ER-positive advanced breast cancer. Importantly, these findings have near-term clinical relevance because PTEN loss also limits the efficacy of PI3Kα inhibitors currently approved in the post-CDK4/6 setting.This article is highlighted in the In This Issue feature, p. 1.

Identification of DHODH as a therapeutic target in small cell lung cancer.

Small cell lung cancer (SCLC) is an aggressive lung cancer subtype with extremely poor prognosis. No targetable genetic driver events have been identified, and the treatment landscape for this disease has remained nearly unchanged for over 30 years. Here, we have taken a CRISPR-based screening approach to identify genetic vulnerabilities in SCLC that may serve as potential therapeutic targets. We used a single-guide RNA (sgRNA) library targeting ~5000 genes deemed to encode "druggable" proteins to perform loss-of-function genetic screens in a panel of cell lines derived from autochthonous genetically engineered mouse models (GEMMs) of SCLC, lung adenocarcinoma (LUAD), and pancreatic ductal adenocarcinoma (PDAC). Cross-cancer analyses allowed us to identify SCLC-selective vulnerabilities. In particular, we observed enhanced sensitivity of SCLC cells toward disruption of the pyrimidine biosynthesis pathway. Pharmacological inhibition of dihydroorotate dehydrogenase (DHODH), a key enzyme in this pathway, reduced the viability of SCLC cells in vitro and strongly suppressed SCLC tumor growth in human patient-derived xenograft (PDX) models and in an autochthonous mouse model. These results indicate that DHODH inhibition may be an approach to treat SCLC.

Author Correction: LKB1 loss links serine metabolism to DNA methylation and tumorigenesis.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

Combination Olaparib and Temozolomide in Relapsed Small-Cell Lung Cancer.

Small-cell lung cancer (SCLC) is an aggressive malignancy in which inhibitors of PARP have modest single-agent activity. We performed a phase I/II trial of combination olaparib tablets and temozolomide (OT) in patients with previously treated SCLC. We established a recommended phase II dose of olaparib 200 mg orally twice daily with temozolomide 75 mg/m2 daily, both on days 1 to 7 of a 21-day cycle, and expanded to a total of 50 patients. The confirmed overall response rate was 41.7% (20/48 evaluable); median progression-free survival was 4.2 months [95% confidence interval (CI), 2.8-5.7]; and median overall survival was 8.5 months (95% CI, 5.1-11.3). Patient-derived xenografts (PDX) from trial patients recapitulated clinical OT responses, enabling a 32-PDX coclinical trial. This revealed a correlation between low basal expression of inflammatory-response genes and cross-resistance to both OT and standard first-line chemotherapy (etoposide/platinum). These results demonstrate a promising new therapeutic strategy in SCLC and uncover a molecular signature of those tumors most likely to respond. SIGNIFICANCE: We demonstrate substantial clinical activity of combination olaparib/temozolomide in relapsed SCLC, revealing a promising new therapeutic strategy for this highly recalcitrant malignancy. Through an integrated coclinical trial in PDXs, we then identify a molecular signature predictive of response to OT, and describe the common molecular features of cross-resistant SCLC.See related commentary by Pacheco and Byers, p. 1340.This article is highlighted in the In This Issue feature, p. 1325.

A Code of Mono-phosphorylation Modulates the Function of RB.

Hyper-phosphorylation of RB controls its interaction with E2F and inhibits its tumor suppressor properties. However, during G1 active RB can be mono-phosphorylated on any one of 14 CDK phosphorylation sites. Here, we used quantitative proteomics to profile protein complexes formed by each mono-phosphorylated RB isoform (mP-RB) and identified the associated transcriptional outputs. The results show that the 14 sites of mono-phosphorylation co-ordinate RB's interactions and confer functional specificity. All 14 mP-RBs interact with E2F/DP proteins, but they provide different shades of E2F regulation. RB mono-phosphorylation at S811, for example, alters RB transcriptional activity by promoting its association with NuRD complexes. The greatest functional differences between mP-RBs are evident beyond the cell cycle machinery. RB mono-phosphorylation at S811 or T826 stimulates the expression of oxidative phosphorylation genes, increasing cellular oxygen consumption. These results indicate that RB activation signals are integrated in a phosphorylation code that determines the diversity of RB activity.

Non-canonical functions of the RB protein in cancer.

The canonical model of RB-mediated tumour suppression developed over the past 30 years is based on the regulation of E2F transcription factors to restrict cell cycle progression. Several additional functions have been proposed for RB, on the basis of which a non-canonical RB pathway can be described. Mechanistically, the non-canonical RB pathway promotes histone modification and regulates chromosome structure in a manner distinct from cell cycle regulation. These functions have implications for chemotherapy response and resistance to targeted anticancer agents. This Opinion offers a framework to guide future studies of RB in basic and clinical research.

E2F/DP Prevents Cell-Cycle Progression in Endocycling Fat Body Cells by Suppressing dATM Expression.

To understand the consequences of the complete elimination of E2F regulation, we profiled the proteome of Drosophila dDP mutants that lack functional E2F/DP complexes. The results uncovered changes in the larval fat body, a differentiated tissue that grows via endocycles. We report an unexpected mechanism of E2F/DP action that promotes quiescence in this tissue. In the fat body, dE2F/dDP limits cell-cycle progression by suppressing DNA damage responses. Loss of dDP upregulates dATM, allowing cells to sense and repair DNA damage and increasing replication of loci that are normally under-replicated in wild-type tissues. Genetic experiments show that ectopic dATM is sufficient to promote DNA synthesis in wild-type fat body cells. Strikingly, reducing dATM levels in dDP-deficient fat bodies restores cell-cycle control, improves tissue morphology, and extends animal development. These results show that, in some cellular contexts, dE2F/dDP-dependent suppression of DNA damage signaling is key for cell-cycle control and needed for normal development.

The metabolic function of cyclin D3-CDK6 kinase in cancer cell survival.

D-type cyclins (D1, D2 and D3) and their associated cyclin-dependent kinases (CDK4 and CDK6) are components of the core cell cycle machinery that drives cell proliferation. Inhibitors of CDK4 and CDK6 are currently being tested in clinical trials for patients with several cancer types, with promising results. Here, using human cancer cells and patient-derived xenografts in mice, we show that the cyclin D3-CDK6 kinase phosphorylates and inhibits the catalytic activity of two key enzymes in the glycolytic pathway, 6-phosphofructokinase and pyruvate kinase M2. This re-directs the glycolytic intermediates into the pentose phosphate (PPP) and serine pathways. Inhibition of cyclin D3-CDK6 in tumour cells reduces flow through the PPP and serine pathways, thereby depleting the antioxidants NADPH and glutathione. This, in turn, increases the levels of reactive oxygen species and causes apoptosis of tumour cells. The pro-survival function of cyclin D-associated kinase operates in tumours expressing high levels of cyclin D3-CDK6 complexes. We propose that measuring the levels of cyclin D3-CDK6 in human cancers might help to identify tumour subsets that undergo cell death and tumour regression upon inhibition of CDK4 and CDK6. Cyclin D3-CDK6, through its ability to link cell cycle and cell metabolism, represents a particularly powerful oncoprotein that affects cancer cells at several levels, and this property can be exploited for anti-cancer therapy.