Predictive Genomic Biomarkers of Hormonal Therapy Versus Chemotherapy Benefit in Metastatic Castration-resistant Prostate Cancer

Drug development; Predictive biomarkers; Prostate cancerDesarrollo de fármacos; Biomarcadores predictivos; Cáncer de próstataDesenvolupament de fàrmacs; Biomarcadors predictius; Càncer de pròstataBackground Biomarkers predicting second-generation novel hormonal therapy (NHT) benefit relative to taxanes are critical for optimized treatment decisions for metastatic castration-resistant prostate cancer (mCRPC) patients. These associations have not been reported simultaneously for common mCRPC genomic biomarkers. Objective To evaluate predictive associations of common genomic aberrations in mCRPC using an established comprehensive genomic profiling (CGP) system. Design, setting, and participants A retrospective cohort study used data from a deidentified US-based clinicogenomic database comprising patients treated in routine clinical practice between 2011 and 2020, evaluated with Foundation Medicine CGP in tissue biopsies obtained around the time of treatment decision. The main cohort included 180 NHT and 179 taxane lines of therapy (LOTs) from 308 unique patients. The sequential cohort comprised a subset of the main cohort NHT LOTs immediately followed by taxane from 55 unique patients. Outcome measurements and statistical analysis Prostate-specific antigen (PSA) response, time to next treatment (TTNT), and overall survival (OS) were assessed. Main cohort analyses were adjusted for known treatment assignment biases via inverse probability of treatment weighting (IPTW) in treatment interaction models. Results and limitations In the main cohort, patients with AR amplification (ARamp) or PTEN aberrations (PTENalt) had worse relative PSA response on NHT versus taxanes compared with patients without. Patients with ARamp, PTENalt, or RB1 aberrations (RB1alt) also had worse relative TTNT and OS on NHT but not on taxanes. In multivariable models for TTNT and OS adjusted via IPTW, ARamp, PTENalt, and RB1alt were shown as poor prognostic factors overall and demonstrated significant treatment interactions, indicating reduced hazards of therapy switch and death on taxanes versus NHT. Consistent associations favoring increased benefit from subsequent taxane despite prior NHT treatment line were observed only for ARamp in the sequential cohort, in which very few patients had RB1alt for assessment. Conclusions ARamp status is a candidate biomarker to predict poor effectiveness of NHT relative to taxanes in mCRPC in scenarios where both options are considered

Merkel cell polyomavirus large T antigen disrupts lysosome clustering by translocating human Vam6p from the cytoplasm to the nucleus

Merkel cell polyomavirus (MCV) has been recently described as the cause for most human Merkel cell carcinomas. MCV is similar to simian virus 40 (SV40) and encodes a nuclear large T (LT) oncoprotein that is usually mutated to eliminate viral replication among tumor-derived MCV. We identified the hVam6p cytoplasmic protein involved in lysosomal processing as a novel interactor with MCV LT but not SV40 LT. hVam6p binds through its clathrin heavy chain homology domain to a unique region of MCV LT adjacent to the retinoblastoma binding site. MCV LT translocates hVam6p to the nucleus, sequestering it from involvement in lysosomal trafficking. A naturally occurring, tumor-derived mutant LT (MCV350) lacking a nuclear localization signal binds hVam6p but fails to inhibit hVam6p-induced lysosomal clustering. MCV has evolved a novel mechanism to target hVam6p that may contribute to viral uncoating or egress through lysosomal processing during virus replication

Bub1 regulates chromosome segregation in a kinetochore-independent manner

The kinetochore-bound protein kinase Bub1 performs two crucial functions during mitosis: it is essential for spindle checkpoint signaling and for correct chromosome alignment. Interestingly, Bub1 mutations are found in cancer tissues and cancer cell lines. Using an isogenic RNA interference complementation system in transformed HeLa cells and untransformed RPE1 cells, we investigate the effect of structural Bub1 mutants on chromosome segregation. We demonstrate that Bub1 regulates mitosis through the same mechanisms in both cell lines, suggesting a common regulatory network. Surprisingly, Bub1 can regulate chromosome segregation in a kinetochore-independent manner, albeit at lower efficiency. Its kinase activity is crucial for chromosome alignment but plays only a minor role in spindle checkpoint signaling. We also identify a novel conserved motif within Bub1 (amino acids 458–476) that is essential for spindle checkpoint signaling but does not regulate chromosome alignment, and we show that several cancer-related Bub1 mutants impair chromosome segregation, suggesting a possible link to tumorigenesis

Cellular and Viral Factors Regulating Merkel Cell Polyomavirus Replication

Merkel cell polyomavirus (MCV), a previously unrecognized component of the human viral skin flora, was discovered as a mutated and clonally-integrated virus inserted into Merkel cell carcinoma (MCC) genomes. We reconstructed a replicating MCV clone (MCV-HF), and then mutated viral sites required for replication or interaction with cellular proteins to examine replication efficiency and viral gene expression. Three days after MCV-HF transfection into 293 cells, although replication is not robust, encapsidated viral DNA and protein can be readily isolated by density gradient centrifugation and typical ∼40 nm diameter polyomavirus virions are identified by electron microscopy. The virus has an orderly gene expression cascade during replication in which large T (LT) and 57kT proteins are first expressed by day 2, followed by expression of small T (sT) and VP1 proteins. VP1 and sT proteins are not detected, and spliced 57kT is markedly diminished, in the replication-defective virus suggesting that early gene splicing and late gene transcription may be dependent on viral DNA replication. MCV replication and encapsidation is increased by overexpression of MCV sT, consistent with sT being a limiting factor during virus replication. Mutation of the MCV LT vacuolar sorting protein hVam6p (Vps39) binding site also enhances MCV replication while exogenous hVam6p overexpression reduces MCV virion production by >90%. Although MCV-HF generates encapsidated wild-type MCV virions, we did not find conditions for persistent transmission to recipient cell lines suggesting that MCV has a highly restricted tropism. These studies identify and highlight the role of polyomavirus DNA replication in viral gene expression and show that viral sT and cellular hVam6p are important factors regulating MCV replication. MCV-HF is a molecular clone that can be readily manipulated to investigate factors affecting MCV replication

PP2A inactivation is a crucial step in triggering apoptin-induced tumor-selective cell killing

Apoptin (apoptosis-inducing protein) harbors tumor-selective characteristics making it a potential safe and effective anticancer agent. Apoptin becomes phosphorylated and induces apoptosis in a large panel of human tumor but not normal cells. Here, we used an in vitro oncogenic transformation assay to explore minimal cellular factors required for the activation of apoptin. Flag-apoptin was introduced into normal fibroblasts together with the transforming SV40 large T antigen (SV40 LT) and SV40 small t antigen (SV40 ST) antigens. We found that nuclear expression of SV40 ST in normal cells was sufficient to induce phosphorylation of apoptin. Mutational analysis showed that mutations disrupting the binding of ST to protein phosphatase 2A (PP2A) counteracted this effect. Knockdown of the ST-interacting PP2A–B56γ subunit in normal fibroblasts mimicked the effect of nuclear ST expression, resulting in induction of apoptin phosphorylation. The same effect was observed upon downregulation of the PP2A–B56δ subunit, which is targeted by protein kinase A (PKA). Apoptin interacts with the PKA-associating protein BCA3/AKIP1, and inhibition of PKA in tumor cells by treatment with H89 increased the phosphorylation of apoptin, whereas the PKA activator cAMP partially reduced it. We infer that inactivation of PP2A, in particular, of the B56γ and B56δ subunits is a crucial step in triggering apoptin-induced tumor-selective cell death

Functional Connection between Rad51 and PML in Homology-Directed Repair

The promyelocytic leukemia protein (PML) is a tumor suppressor critical for formation of nuclear bodies (NBs) performing important functions in transcription, apoptosis, DNA repair and antiviral responses. Earlier studies demonstrated that simian virus 40 (SV40) initiates replication near PML NBs. Here we show that PML knockdown inhibits viral replication in vivo, thus indicating a positive role of PML early in infection. SV40 large T antigen (LT) induces DNA damage and, consequently, nuclear foci of the key homologous recombination repair protein Rad51 that colocalize with PML. PML depletion abrogates LT-induced Rad51 foci. LT may target PML NBs to gain access to DNA repair factors like Rad51 that are required for viral replication. We have used the SV40 model to gain insight to DNA repair events involving PML. Strikingly, even in normal cells devoid of viral oncoproteins, PML is found to be instrumental for foci of Rad51, Mre11 and BRCA1, as well as homology-directed repair after double-strand break (DSB) induction. Following LT expression or external DNA damage, PML associates with Rad51. PML depletion also causes a loss of RPA foci following γ-irradiation, suggesting that PML is required for processing of DSBs. Immunofluorescent detection of incorporated BrdU without prior denaturation indicates a failure to generate ssDNA foci in PML knockdown cells upon γ-irradiation. Consistent with the lack of RPA and BrdU foci, γ-irradiation fails to induce Chk1 activation, when PML is depleted. Taken together, we have discovered a novel functional connection between PML and the homologous recombination-mediated repair machinery, which might contribute to PML tumor suppressor activity

Molecular residual disease detection in resected, muscle-invasive urothelial cancer with a tissue-based comprehensive genomic profiling–informed personalized monitoring assay

IntroductionCirculating tumor DNA (ctDNA) detection postoperatively may identify patients with urothelial cancer at a high risk of relapse. Pragmatic tools building off clinical tumor next-generation sequencing (NGS) platforms could have the potential to increase assay accessibility.MethodsWe evaluated the widely available Foundation Medicine comprehensive genomic profiling (CGP) platform as a source of variants for tracking of ctDNA when analyzing residual samples from IMvigor010 (ClinicalTrials.gov identifier NCT02450331), a randomized adjuvant study comparing atezolizumab with observation after bladder cancer surgery. Current methods often involve germline sampling, which is not always feasible or practical. Rather than performing white blood cell sequencing to filter germline and clonal hematopoiesis (CH) variants, we applied a bioinformatic approach to select tumor (non-germline/CH) variants for molecular residual disease detection. Tissue-informed personalized multiplex polymerase chain reaction–NGS assay was used to detect ctDNA postsurgically (Natera).ResultsAcross 396 analyzed patients, prevalence of potentially actionable alterations was comparable with the expected prevalence in advanced disease (13% FGFR2/3, 20% PIK3CA, 13% ERBB2, and 37% with elevated tumor mutational burden ≥10 mutations/megabase). In the observation arm, 66 of the 184 (36%) ctDNA-positive patients had shorter disease-free survival [DFS; hazard ratio (HR) = 5.77; 95% confidence interval (CI), 3.84–8.67; P < 0.0001] and overall survival (OS; HR = 5.81; 95% CI, 3.41–9.91; P < 0.0001) compared with ctDNA-negative patients. ctDNA-positive patients had improved DFS and OS with atezolizumab compared with those in observation (DFS HR = 0.56; 95% CI, 0.38–0.83; P = 0.003; OS HR = 0.66; 95% CI, 0.42–1.05). Clinical sensitivity and specificity for detection of postsurgical recurrence were 58% (60/103) and 93% (75/81), respectively.ConclusionWe present a personalized ctDNA monitoring assay utilizing tissue-based FoundationOne® CDx CGP, which is a pragmatic and potentially clinically scalable method that can detect low levels of residual ctDNA in patients with resected, muscle-invasive bladder cancer without germline sampling