Multivariable analysis for overall survival and relapse-free survival demonstrating lack of significant for D14 BM status. The most recent National Comprehensive Cancer Network guidelines for acute myeloid leukemia (AML) patients undergoing cytarabine-based induction therapy recommend the performance of a bone marrow biopsy 14–21 days after the initiation of therapy.1 These "Day 14" bone marrow biopsies (D14 BM Bx) are routinely performed to assess early response to induction therapy. The presence of residual disease (RD) has been historically associated with decreased rates of complete remission (CR) at count recovery and has been utilized to justify the pursuit of prompt reinduction therapy. In recent years, both the prognostic value of D14 BM Bx and the clinical utility of reinduction decisions based on its findings have been called into question. The limitations have centered on the practice's limited specificity for predicting CR, with several studies suggesting that up to one third of patients with RD at D14 can go on to achieve CR without reinduction therapy.2, 3 There is still a paucity of randomized clinical trial data to indicate whether reinduction for D14 RD confers benefit to major clinical endpoints such as overall survival (OS) and relapse-free survival (RFS). D14 BM Bx nonetheless remains one of the few posttreatment variables available to guide decisions regarding the course of induction therapy. Although its limitations in isolation are well appreciated, it remains unclear whether the degree of chemoresistance reflected in D14 RD independently informs the clinical heterogeneity still observed in other widely validated prognostic criteria such as European LeukemiaNet (ELN) risk classification.4 To explore this question, we retrospectively identified adult patients with AML who received intensive induction therapy with a "7 + 3" (cytarabine and an anthracycline) regimen and D14 BM Bx at Mount Sinai Hospital from January 2009 to July 2022 and analyzed the relationship between D14 disease status, 2022 ELN risk classification, and receipt of reinduction therapy on major clinical endpoints. D14 RD was defined as an elevated marrow cellularity >20% and/or blast counts >5%. Patients were classified according to the 2022 ELN risk stratification guidelines. For all survival analyses, patients were censored at last follow-up, death, or time of stem cell transplantation (SCT). The baseline characteristics of our cohort are outlined in Table S1. The study population consisted of 200 patients with a median age of 57 years and 52.7% were female. Most patients had de novo AML (76%). Patients' risk groups were classified based on 2022 ELN guidelines (19% favorable, 49% intermediate, 29% adverse, 4% not available). In total, 113 patients (57%) had no evidence of disease (NED) at D14 and 87 patients (43%) had RD. When comparing baseline characteristics, patients with NED were more likely to have favorable ELN risk group (p = .01) than those with RD; however, there were no significant differences in age, sex, race/ethnicity, presenting lab values, type of AML (primary vs. secondary), or diagnostic bone marrow biopsy parameters. Rates of CR at count recovery were similar between those with NED versus RD (85% vs. 78%; p = .22), and a multivariable model for predictors of CR showed only adverse ELN risk to have a statistically significant independent relationship with achievement of CR (OR = 0.145; p = .02; Table S2). Among those with RD, 56 patients (64%) received reinduction therapy. Of those 46 patients (82%) were treated with an intensive regimen and 10 patients (18%) were treated with a non-intensive regimen (Table S3). Patients with RD who received reinduction had higher rates of intermediate/adverse risk AML (p = .01) and higher median D14 blast percentage (36% vs. 6%, p < .01) and cellularity (30% vs. 20%, p = .04) compared with those who did not. Those who received reinduction therapy were more likely to achieve a CR (86% vs. 63% without; p = .02). There was no statistically significant difference in median OS between patients with NED and RD (3.8 vs. 1.4 years; p = .24). Among patients who achieved CR, those with NED had an increased RFS (2.6 vs. 0.8 years; p = .01). Within individual ELN risk classes, we did not observe a difference in OS when stratified by D14 disease status. Among patients with intermediate risk disease, those with NED at D14 had a longer RFS (2.6 vs. 0.5 years with RD; p = .03). Neither of the aforementioned RFS differences remained statistically significant when censoring at time of SCT was not performed (p = .24 and .22, respectively). There was no difference in RFS among patients with favorable or adverse risk disease when stratified by D14 disease status. A summary of these clinical endpoints can be found in Table S4, and pertinent survival analyses are depicted in Figures S1–S3. Among patients with RD, those who received reinduction therapy trended toward decreased OS (1.2 years vs. nonestimable [NE]; p = .15) and had decreased RFS (0.4 vs. 1.2 years; p = .02) relative to those who did not. When not censoring at time of SCT, this difference in RFS was no longer statistically significant (1.0 years vs. NE; p = .13). When comparing those with NED and those with RD who did not receive reinduction therapy, there was no difference in OS (p = .55) or RFS (p = .73). In the unadjusted Cox regression model, OS was not significantly affected by D14 BM status (hazard ratio [HR] = 1.3; p = .28). In those with RD, reinduction was associated with an increased hazard of all-cause mortality (HR = 2.2; p = .0004). The multivariable OS model (Table 1) showed no evidence of higher mortality risk among patients with RD (vs. NED, HR = 0.98; p = .97) or those with RD who received reinduction therapy (vs. RD without; HR = 1.9; p = .18). In the unadjusted Cox regression model, hazard of relapse was significantly increased among patients with RD (vs. NED; HR = 1.9; p = .02). In those with RD, reinduction was associated with an increased hazard of relapse (vs. no reinduction; HR = 2.8; p < .01). The multivariable RFS model showed no difference in hazard of relapse regardless of D14 disease or, among those with RD, receipt of reinduction therapy (Table 1). The clinical role of D14 BM Bx remains unclear, and these findings further question this routine practice by showing similar clinical outcomes irrespective of D14 disease status. Our cohort showed similar OS and rates of CR regardless of D14 disease status. Although those with NED showed decrease hazard of relapse on univariable analysis, this relationship was not seen on multivariable analysis. Additionally, while those with RD were more likely to have intermediate or adverse ELN risk disease, there was no major difference in median OS or RFS within these groups when stratifying by D14 disease status save for an increased RFS among patients with intermediate risk disease and NED at D14. Given that this relationship did not hold true when censoring at time of SCT was deferred in the survival analysis, it suggests that clinicians may have been motivated to pursue expeditious SCT in intermediate risk patients with RD. Overall, these findings suggest that D14 RD is more likely a reflection of underlying disease severity rather than an independent variable that can further refine the clinical heterogeneity still observed within individual ELN risk groups. Our findings also raise questions regarding the benefit of reinduction therapy for D14 RD. There were increased rates of CR among those with RD who received reinduction compared with those who did not—rates comparable to those seen in patients with NED at D14. However, these impressive rates of CR did not reliably extrapolate to long-term clinical endpoints. There was a trend toward decreased OS and a statistically significant decrease in RFS among those with RD who received reinduction therapy compared with those with NED and those with RD who did not receive reinduction therapy. Survival analysis also showed no significant difference in median OS or RFS between patients with NED at D14 and those who had RD but did not receive reinduction therapy. Interpretation of these findings is limited given that higher relative blast counts at D14 likely influenced the decision to provide reinduction therapy to patients. Most patients with RD who did not receive reinduction therapy were composed of patients with high marrow cellularity and low blast percentage on D14—sometimes referred to as an "indeterminate disease" category that has previously shown similar outcomes with reinduction or observation.5 It is possible that reinduction therapy may have exerted a salvage effect on these patients with a higher burden of RD that may have increased rates of CR and, in turn, improved long-term clinical endpoints compared with what outcomes would have been in the absence of reinduction therapy. Such a salvage effect alludes to prior studies that have shown similar long-term outcomes among patients who achieve CR regardless of whether they did so with one or two induction cycles.6 Given the retrospective nature of our study, our findings are limited by selection bias and had a disproportionate focus on patients receiving reinduction with intensive regimens. Use of additional induction agents beyond cytarabine and an anthracycline was also not uniformly documented in our cohort. Nonetheless, replication of these analyses with larger cohorts and in a prospective randomized trial format is merited to more robustly evaluate the prognostic and clinical value of D14 BM Bx as AML risk stratification models and therapeutic options continue to evolve. JM receives research funding paid to the institution from Incyte, Novartis, Celgene, BMS, Kartos, Karyopharm, PharmaEssentia, Abbvie, Geron, CTI Bio, and consulting fees from Incyte, Kartos, Karyopharm, Geron, Roche, Abbvie, CTI Bio, GSK, Pfizer, PharmaEssentia, Galecto, Celgene, BMS, and Novartis. DT receives contracted research funding paid to his institution from CTI Biopharma, Astellas Pharma, and Gilead and consulting fees from CTI Biopharma, Novartis, AbbVie, Sierra Oncology, GSK, and Cogent Biosciences. JF receives contracted research funding paid to his institution from Syros and Oryzon pharmaceuticals. MK receives research funding paid to the institution from Incyte, Celgene, BMS, Morphosys, Protagonist, Ionis, Silence therapeutics, Kura oncology and consulting fees from Incyte, Abbvie, Morphosys, and Protagonist. LS has served on advisory boards for Jazz Pharmaceuticals, Servier, and Beam Therapeutics. The remaining authors have no conflicts of interest to report. As a retrospective study, no consent was obtained from subjects in accordance with a waiver obtained from the Program for the Protection of Human Subjects at the Icahn School of Medicine at Mount Sinai. Table S1. Baseline characteristics of overall cohort. Table S2A. Univariable model for complete remission. Table S2B. Multivariable model for complete remission. Table S3. Reinduction regimens utilized. Table S4. Summary of clinical endpoint by presence of residual disease at Day 14 and receipt of reinduction therapy. Figure S1. (A) Overall survival stratified by residual disease at Day 14. (B) Relapse-free survival stratified by residual disease at Day 14. Figure S2. (A) Overall survival stratified by presence of residual disease at Day 14 and receipt of reinduction therapy. (B) Relapse-free survival stratified by presence of residual disease at Day 14 and receipt of reinduction therapy. Figure S3. Kaplan–Meier curves of overall and relapse-free survival stratified by presence of residual disease at D14 among each ELN risk group. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.
7555 Background: Myeloma cast nephropathy (MCN) has been a well-known negative predictive marker in newly diagnosed multiple myeloma (NDMM). Anti-CD38 + monoclonal antibodies (mAb) have improved outcomes, but little is known about their impact in patients with MCN. We performed a retrospective cohort study to investigate the outcomes of patients with MCN in the era of anti-CD38 + mAb-based frontline therapy. Methods: 115 NDDM patients received frontline anti-CD38 + mAb from 11/15/18 to 1/24/23. MCN was defined as evidence of light chain casts on biopsy, serum creatinine of >2mg/dL, or 2021 CKD-EPI eGFR of <40mL/min/1.73m 2 ; ≥1g/d proteinuria; and involved FLC ≥50mg/dL. 23 had MCN; 92 were contemporary controls. We obtained data regarding clinical course. Results: 6 MCN patients needed hemodialysis (HD) at diagnosis. MCN patients had similar R2-ISS to controls. Median proteinuria in MCN patients was 5.7g/d more than in controls; serum creatinine (Cr) was 3.2mg/dL higher; and median hemoglobin was 1.5g/dL less. More MCN patients received quadruplet regimens (86.9% vs 62.0%, p = 0.010); similar proportions were transplanted. At 1, 3, and 6 months, serum Cr was higher in MCN patients (p < 0.001, p = 0.002, p = 0.002). Urine protein:creatinine ratios trended toward difference at 1 month (2.04 vs 0.15g/g MCN vs controls, p = 0.061) but were similar at 3 and 6 months. Response rates at 6 months did not differ. 4 MCN patients still required HD after 6 months. Median follow-up was 23.0-23.5 months. 4 MCN patients died, 1 due to progressive disease. No significant difference in disease-free survival (DFS; HR = 0.58, 95% CI 0.20-1.64, p = 0.295), time to next treatment (TTNT; HR = 0.53, 95% CI 0.20-1.39, p = 0.191), or overall survival (OS; HR 0.610, 95% CI 0.19-1.98, p = 0.406) was seen at any point between MCN and control cohorts. 1 year DFS, TTNT, and OS were: 87.0% MCN, 94.5% control; 77.2% MCN, 91.7% control; 87.9% MCN, 96.1% control. By statistical equivalence testing, MCN DFS and TTNT were within 6 months of control DFS and TTNT (p = 0.038, p = 0.032); MCN OS was within 5 months of control OS (p = 0.034). Conclusions: Patients with NDMM and MCN who receive upfront anti-CD38 + mAb therapy experience prolonged survival compared to prior findings. Anti-CD38 + mAb may substantially reduce, though not fully eliminate, the negative survival impact of MCN. Multiple mechanisms, including time to proteinuria resolution, may be involved. Further study is warranted. [Table: see text]
Abstract The objective of our study was to report real-world data on the safety and efficacy of standard-of-care teclistamab in patients with relapsed/refractory multiple myeloma (MM). This is a multi-institutional retrospective cohort study and included all consecutive patients that received at least one dose of teclistamab up until August 2023. One hundred and ten patients were included, of whom, 86% had triple-class refractory disease, 76% penta-refractory disease, and 35% had prior exposure to B-cell maturation antigen (BCMA)-targeting therapies. The overall response rate (ORR) in our cohort was 62%, with a ≥ very good partial remission (VGPR) rate of 51%. The ORR in patients with and without prior BCMA-targeted therapies was 54% vs 67%, respectively ( p = 0.23). At a median follow-up of 3.5 months (range, 0.39–10.92), the estimated 3 month and 6 month progression free survival (PFS) was 57% (95% CI, 48%, 68%) and 52% (95% CI, 42%, 64%) respectively. The incidence of cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS) was 56% and 11% respectively, with grade ≥3 CRS and ICANS noted in 3.5% and 4.6% of patients respectively. 78 unique infections were diagnosed in 44 patients, with the incidence of all-grade and grade ≥3 infections being 40% vs 26% respectively. Primary prophylaxis with intravenous immunoglobulin (IVIG) was associated with a significantly lower infection risk on multivariate analysis (Hazard ratio [HR] 0.33; 95% CI 0.17, 0.64 ; p = 0.001).
Resistance to regeneration of insulin-producing pancreatic β cells is a fundamental challenge for type 1 and type 2 diabetes. Recently, small molecule inhibitors of the kinase DYRK1A have proven effective in inducing adult human β cells to proliferate, but their detailed mechanism of action is incompletely understood. We interrogated our human insulinoma and β cell transcriptomic databases seeking to understand why β cells in insulinomas proliferate, while normal β cells do not. This search reveals the DREAM complex as a central regulator of quiescence in human β cells. The DREAM complex consists of a module of transcriptionally repressive proteins that assemble in response to DYRK1A kinase activity, thereby inducing and maintaining cellular quiescence. In the absence of DYRK1A, DREAM subunits reassemble into the pro-proliferative MMB complex. Here, we demonstrate that small molecule DYRK1A inhibitors induce human β cells to replicate by converting the repressive DREAM complex to its pro-proliferative MMB conformation.
Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The restricted host tropism of hepatitis C virus (HCV) remains incompletely understood, especially post-entry, and has hindered developing an immunocompetent, small animal model. HCV replication in non-permissive species may be limited by incompatibilities between the viral replication machinery and orthologs of essential host factors, like cyclophilin A (CypA). We thus compared the ability of CypA from mouse, tree shrew, and seven non-human primate species to support HCV replication, finding that murine CypA only partially rescued viral replication in Huh7.5-shRNA CypA cells. We determined the specific amino acid differences responsible and generated mutants able to fully rescue replication. We expressed these mutants in engineered murine hepatoma cells and although we observed increases in HCV replication following infection, they remained far lower than those in highly permissive human hepatoma cells, and minimal infectious particle release was observed. Together, these data suggest additional co-factors remain unidentified. Future work to determine such factors will be critical for developing an immunocompetent mouse model supporting HCV replication. https://doi.org/10.7554/eLife.44436.001 eLife digest Hepatitis C is a life-long disease that begins when a virus infects the cells of the liver. Although the infection is curable, it is expensive to treat, and there is not yet a vaccine to prevent the disease. This is largely because the virus that causes hepatitis C, also known as HCV, naturally only infects humans and chimpanzees, which has made it difficult to generate an effective animal model for developing a vaccine. Mice are frequently used as a model for studying disease and can be genetically altered to allow HCV to enter their liver cells. However, once HCV enters mouse cells, it struggles to replicate. As a result, an infection does not develop, and the immune system's response to the virus cannot be fully explored. Replication of HCV in humans relies on a protein called cyclophilin A, or CypA for short. Now, Gaska et al. have set out to improve current animal models for HCV by investigating whether HCV can also use CypA from other species, including mice, to replicate. Gaska et al. showed that the mouse form of CypA could help HCV replicate in human liver cells with lower than normal levels of CypA, but only very poorly. Editing the mouse gene for CypA to be more like the human version resulted in higher HCV replication. Putting variants of CypA into the liver cells of mice, which do not normally replicate HCV at high levels, led to an increase in HCV replication. However, replication of HCV in mice was still far lower than in human liver cells, suggesting that the mouse model system could be improved by learning more about which proteins interact with CypA. Injection drug use – one of the main ways hepatitis C spreads – is becoming increasingly common because of the growing opioid epidemic in many countries. A clinically relevant animal model that supports hepatitis C virus infection would be an important milestone towards a vaccine that could prevent the continued spread of this disease. https://doi.org/10.7554/eLife.44436.002 Introduction Every year, an estimated 3–4 million individuals become newly infected with hepatitis C virus (HCV) (Westbrook and Dusheiko, 2014), with 60–80% going on to join the population of approximately 71.1 million who are chronically infected (Blach et al., 2017). A hepatotropic virus, HCV has only been shown to robustly infect in vivo human and chimpanzee hepatocytes. This limited host tropism has proven problematic in developing an animal model that is not only ethically and financially sound but also immunocompetent. Such a model would allow the study of the underlying immunopathogenesis of HCV infection as well as the development and testing of vaccine candidates. Most efforts to generate such an in vivo model have focused on mice, which are amenable to genetic manipulation and have many well-established research tools built around their use. Overcoming the natural imperviousness of murine hepatocytes to HCV has required adjustments at multiple stages of the virus life cycle. Barriers at the level of entry (McCaffrey et al., 2002; Park et al., 2009) in murine cells could not be overcome until the identification of the four canonical HCV human entry factors – claudin-1 (CLDN1; Evans et al., 2007), occludin (OCLN; Liu et al., 2009a; Ploss et al., 2009), CD81 (Pileri et al., 1998) and scavenger receptor class B member 1 (SCARB1; Scarselli et al., 2002). Of these, human CD81 and human OCLN were the minimal factors needed for viral entry (Ploss et al., 2009). Alternatively, infecting with HCV adapted to utilize murine CD81 could also successfully overcome this initial obstacle (Bitzegeio et al., 2010). Once inside murine cells, HCV faces another block at the level of replication, which initial studies circumvented through the use of selectable subgenomic HCV replicons (McCaffrey et al., 2002; Park et al., 2009; Uprichard et al., 2006). Murine cells did appear to support assembly and release of infectious particles, albeit at low levels, once NS2 and the structural HCV proteins were provided in trans and the murine or human ortholog of the apolipoprotein ApoE was overexpressed (Long et al., 2011). Further efforts to improve HCV replication in a murine context have relied on disruption of innate immune responses (Aly et al., 2011; Anggakusuma et al., 2015; Chang et al., 2006; Frentzen et al., 2014; Lin et al., 2010; Nandakumar et al., 2013), with robust replication and completion of the HCV life cycle proving difficult following infection unless a selectable genome is used (Vogt et al., 2013). These poor levels of replication could be due to the lower compatibility of murine orthologs of vital replication host factors with the viral replication machinery or the absence of proteins that normally facilitate these interactions in human cells. Although numerous intracellular host factors have been implicated in HCV replication, extensive experimental evidence has only been provided for three host factors: cyclophilin A (CypA) (Kaul et al., 2009; Yang et al., 2008a), phosphatidylinositol four kinase IIIα (PI4KA) (Berger et al., 2009; Borawski et al., 2009; Reiss et al., 2011; Tai et al., 2009; Trotard et al., 2009) and microRNA-122 (miR-122) (Jopling et al., 2005; Lanford et al., 2010; Machlin et al., 2011). miR-122 is highly conserved between humans and other species, including mice, making its expression alone unlikely to explain the weak replication of HCV in murine cells. In an effort to systemically dissect the impact of such replication co-factors during infection, we focused in this study on CypA and how it may contribute to the restricted host range of HCV. CypA is a cytosolic 18 kDa peptidyl-prolyl cis-trans isomerase (PPIase) and a part of the biologically ubiquitous cyclophilin enzyme family (Fischer et al., 1989), the members of which were first characterized in mammals by their common ability to bind the immunosuppressive drug cyclosporin A (CsA) and their shared cyclophilin-like domain (CLD) which catalyzes the cis-trans isomerization of proline residues (reviewed in Marks, 1996). CypA overexpression has been implicated in a wide variety of human diseases, ranging from cancer to atherosclerosis (reviewed in Nigro et al., 2013), and it has a demonstrated role in the life cycles of multiple viruses besides HCV (de Wilde et al., 2018; Frausto et al., 2013; Li et al., 2016; Phillips et al., 2015; Tian et al., 2010; von Hahn and Ciesek, 2015; Watashi and Shimotohno, 2007; Zhou et al., 2012). Early work showed that CsA had an inhibitory effect on HCV in chronically infected chimpanzees, but it was not until subsequent in vitro CypA knockdown experiments and dose-response assays with CsA derivatives that CypA was specifically recognized as critical to HCV replication (Chatterji et al., 2009; Ciesek et al., 2009; Coelmont et al., 2009; Kaul et al., 2009; Liu et al., 2009b; Yang et al., 2008b). These studies showed that CypA's relevance to HCV replication was intimately linked to its PPIase activity, as the introduction of point mutations in the PPIase active site led to impaired viral replication (Chatterji et al., 2009; Kaul et al., 2009; Liu et al., 2009b). Individuals exhibiting an HCV non-permissive phenotype were shown to express a rare homozygosity at any of three SNP sites in the coding region of CypA – but not in the enzymatic active site – that subsequent in vitro work showed resulted in markedly decreased levels of intracellular CypA (von Hahn et al., 2012). Despite its known importance, the exact mechanism by which CypA facilitates HCV replication remains poorly characterized. Interactions between CypA and several HCV proteins have been demonstrated, including the RNA-dependent RNA polymerase (RdRP) NS5B (Chatterji et al., 2009; Fernandes et al., 2007; Robida et al., 2007; Yang et al., 2008b), NS5A (Anderson et al., 2011; Coelmont et al., 2010; Foster et al., 2011; Grisé et al., 2012; Hanoulle et al., 2009; Nag et al., 2012; Verdegem et al., 2011) and NS2 (Ciesek et al., 2009; Kaul et al., 2009), but how CypA's binding, PPIase activity and the viral polyprotein are precisely intertwined remains to be understood. The specific impact that cross-species differences in CypA might have on HCV replication and the restricted host tropism of this virus remains an open question and one the present study sought to address. Here, we examined the ability of CypA from diverse species, some of which could serve as feasible small animal models for HCV, to facilitate HCV replication. We found that murine CypA, relative to human CypA, is less proficient at facilitating HCV replication due to differences at the amino acid level and that overexpression of human CypA can increase replication in an engineered murine hepatoma line. Results Ability of diverse CypA orthologs to facilitate HCV replication Knowing the critical role of human CypA in facilitating HCV replication, we first examined the conservation of CypA at the amino acid level across diverse species, focusing on those with promise to serve as biomedical research models and/or closely related to humans. As observed in mice, previous in vivo studies have suggested that several non-human primate (NHP) species – including cynomolgus, Japanese, and rhesus macaque; African green monkey; and Chacma and doguera baboons – appear resistant to HCV infection (Abe et al., 1993; Bukh et al., 2001). In contrast, more recent work in vitro demonstrated that primary hepatocytes from rhesus macaques (PRMH) (Scull et al., 2015) as well as hepatocyte-like cells derived from pigtailed macaque induced pluripotent stem cells (iPSCs) could support the HCV life cycle (Sourisseau et al., 2013). Importantly, pharmacological-mediated suppression of innate immune responses via Jak inhibition enhanced viral replication in PRMH (Scull et al., 2015). Additionally, albeit with limited evidence, tree shrews have also been demonstrated as a potential platform for studying HCV infection (Amako et al., 2010; Tong et al., 2011; Xie et al., 1998; Xu et al., 2007). Thus, in the present study we compared the amino acid similarity of CypA from great apes (human, chimpanzee, bonobo, gorilla, orangutan), Old World monkeys (rhesus macaque, pigtailed macaque, olive baboon), a New World monkey (squirrel monkey), tree shrew, and mouse (Figure 1a). Human, chimpanzee, bonobo, gorilla, olive baboon, and rhesus macaque CypA are 100% identical at the amino acid level and for subsequent experiments the human CypA (hCypA) CDS was used as the representative sequence for these six species. Multiple studies have shown that pigtailed macaques are predominantly homozygous for an insertion of the CypA exon at the TRIM5 locus, resulting in a chimeric TRIM5-CypA transcript (Brennan et al., 2008; Liao et al., 2007; Newman et al., 2008), which we used for our experiments. Figure 1 with 2 supplements see all Download asset Open asset Murine CypA has a diminished ability to facilitate HCV replication. (A) An amino acid sequence alignment of CypA from diverse species. Similar amino acids are indicated in boxed, bold capital letters while differences are lowercase. Species are arranged from top to bottom in increasing evolutionary distance from human. For pigtailed macaque, all experiments utilized a TRIM5-CypA fusion – only the residues of the CypA portion of the fusion are depicted here. (B) Huh7.5 cells expressing an shRNA against endogenous human CypA (Huh7.5-shRNA CypA) were transduced to express different CypA orthologs and then infected with a HCV reporter genome expressing secreted Gaussia luciferase (Jc1-Gluc, MOI = 0.1). At five dpi, the luciferase activity of the supernatants was assessed as a proxy for viral replication. CypA rescue efficiency is shown normalized to Huh7.5-shRNA CypA transduced with human CypA, which is 100% identical at the amino acid level to chimpanzee, bonobo, gorilla, olive baboon and rhesus macaque CypA. Results shown are from two representative experiments, each with triplicate samples. Lines and error bars represent the mean ± SD. Ordinary two-way ANOVA test performed followed by Dunnett's multiple comparisons test with all means compared to that of the +human CypA line. Chimp/Ch, chimpanzee; Bo, bonobo; Go, gorilla; Orang, orangutan; Rhesus mac/RhM, rhesus macaque; Pt mac, pigtailed macaque; Olive bab/Bab, olive baboon; Sq monkey, squirrel monkey. ****, p<0.0001; ns, not significant. https://doi.org/10.7554/eLife.44436.003 Having identified these differences between CypA orthologs, we then compared their respective abilities to support HCV replication in a Huh7.5 cell line stably expressing an shRNA against endogenous human CypA (Huh7.5-shRNA CypA) (von Hahn et al., 2012) (Figure 1—figure supplement 1). Cells were transduced with a bicistronic lentivirus to express the CypA ortholog and a GFP-ubiquitin-neomycin resistance (GUN) fusion protein. The bicistronically expressed GFP provides a straightforward means to monitor protein expression indirectly. We deliberately chose not to add an epitope tag onto the different CypA variants to avoid impacting function. The percentage of GFP+ cells as determined by flow cytometry indicated >60% transduction efficiency (Figure 1—figure supplement 2a). We also assessed protein expression by western blot using two different antibodies with different CypA antigen specificities – commercial antibodies with listed reactivity for human and mouse CypA were available but not for any of the other species under examination (Figure 1—figure supplement 2b, Supplementary file 1). All the orthologs were readily detected at the expected size of ~17 kDa except for squirrel monkey CypA, the signal for which was <2 times that of the background levels in the nontransduced Huh7.5-shRNA CypA cells, and pigtailed macaque CypA. In the latter case, the TRIM5-CypA fusion protein was expected at ~51 kDa but no signal was observed with either antibody. The transduced cells were subsequently infected with the HCV reporter virus Jc1-Gluc (Marukian et al., 2008) at an MOI of 0.1. Levels of Gaussia luciferase in the culture supernatants were thus used as a proxy for assessing HCV replication to compare the rescue efficiencies of the CypA orthologs. As expected, expression of human CypA in Huh7.5-shRNA CypA cells increased HCV replication by more than two logs relative to the non-rescued cells at five days post-infection (dpi) (Figure 1b). Of the orthologs tested, only orangutan CypA, which differs from human CypA by a single amino acid, was capable of rescuing HCV replication at levels similar to human. Compared to human CypA (normalized to 100%), mouse CypA could still facilitate HCV replication but at levels ~3–4% of those observed for human, that is ca. 30-fold decrease. Tupaia CypA, which was well expressed by western blot, did not significantly increase HCV replication above the levels observed in the parental Huh7.5-shRNA CypA cells. It remains possible that the lack of HCV replication in cells transduced with squirrel monkey and pigtailed macaque CypA is due to these proteins not being properly expressed. However, as the transduction efficiency of our constructs expressing pigtailed macaque and squirrel monkey CypA was robust and we were able to detect all other CypA variants utilized in this study by western blot, it is more likely that the antibody reactivity for these two specific orthologs is weaker. Identifying the amino acid basis for the decreased ability of mouse CypA to facilitate HCV replication As known blocks in the viral life cycle at the level of entry have been well characterized in murine hepatocytes, we aimed to further understand how murine CypA might affect viral replication. Thus, still in a human context, we sought to determine how the six amino acid differences between murine and human CypA contributed to the decreased ability of murine CypA to facilitate HCV replication in Huh7.5-shRNA CypA cells. 'Murinized' human CypA and 'humanized' murine CypA mutants were generated whereby each of the six differing amino acids were changed one at a time to their murine or their human counterpart, respectively (Figure 2a), and transduction (Figure 2—figure supplement 1) as well as CypA expression confirmed (Figure 2—figure supplement 2, Supplementary file 1). None of these differences fell in the CsA-binding site of CypA (Figure 2b). Human CypA was more sensitive to changes in the amino acid sequence, with significant decreases in rescue efficiency for all single residue changes tested (Figure 2c). Mouse CypA demonstrated a greater ability to facilitate HCV replication, at least in a human cell context, when either residues 12, 14, 52 or 76 were altered, with levels of replication comparable to those observed in the presence of human CypA (Figure 2d). As three of the residues that differ between mouse and human CypA are clustered together (residues 11, 12 and 14), we also constructed and tested mutants triply 'humanized' or 'murinized' at these positions. Indeed, we observed a striking reversal of phenotype for both constructs, with 'humanized' murine CypA able to rescue HCV replication at levels comparable to human and vice versa for 'murinized' human CypA (Figure 2e). Figure 2 with 2 supplements see all Download asset Open asset Characterizing the amino acid basis for the differing efficiencies of murine and human CypA in HCV replication. (A) Schematic depicting the humanized murine CypA and murinized human CypA constructs tested. (B) Modeled structure of human CypA (PDB 1CWA) with the six residues differing between murine and human CypA altered to those of murine CypA and shown labeled. The residues that directly interact with cyclosporine A (CsA) (Arg55, Phe60, Met61, Gln63, Gly72, Ala101, Asn102, Ala103, Gln111, Phe113, Trp121, Leu122 and His126) (Ke et al., 1994) are depicted in gray as stick models. The six residues that comprise the active site (His54, Arg55, Phe60, Gln111, Phe113, and His126) (Zydowsky et al., 1992), five of which also interact with CsA, are shown in black as stick models. Huh7.5-shRNA CypA cells were transduced with the singly murinized human (C), the singly humanized murine (D) or the triply murinized/humanized (E) CypA mutants, infected with Jc1-Gluc at MOI = 0.1 and supernatants assessed for Gaussia luciferase activity as a proxy for HCV replication at five dpi. The rescue efficiency of each mutant was normalized to Huh7.5-shRNA CypA cells transduced with human CypA. Results shown are from two representative experiments, each with triplicate samples. Lines and error bars represent the mean ± SD. Ordinary two-way ANOVA test performed followed by Sidak's multiple comparison test, with all means compared to that of the +human CypA line (C) or the +mouse CypA line (D). For (E), Tukey's multiple comparison test was used to compare all the means to one another. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001; ns, not significant. https://doi.org/10.7554/eLife.44436.006 Since humanizing residue 52 in murine CypA also resulted in a strong and significant increase in HCV replication, we combined the S52C mutant with each of the individual mutants T11A, A12V, and D14G as well as with the triply humanized mouse mutant T11A/A12V/D14G to ascertain whether there was an additional increase in HCV replication (Figure 3a, Figure 3—figure supplement 1, Supplementary file 1). Mutating residue 52, even in the triple mutant, did not have a significant synergistic effect for any of the mutants tested (Figure 3b). Compared to mutant T11A, mutant T11A/S52C did demonstrate increased rescue efficiency, but upon performing further statistical tests to those shown in the figure, this was not statistically significant. Figure 3 with 1 supplement see all Download asset Open asset Humanizing residue 52 in the murine CypA mutant T11A/A12V/D14G does not further increase rescue efficiency. (A) Schematic of the additional humanized mouse CypA mutants tested. (B) Huh7.5-shRNA CypA cells were transduced with the mutants shown in (A) and infected with Jc1-Gluc at MOI = 0.1. Supernatants were assessed for Gaussia luciferase activity as a proxy for HCV replication at five dpi, and the rescue efficiency of each mutant was normalized to Huh7.5-shRNA CypA transduced with human CypA. Results shown are from two representative experiments, each with triplicate samples. Lines and error bars represent the mean ± SD. Ordinary two-way ANOVA test performed followed by Sidak's multiple comparison test, with all means compared to that of the +mouse CypA line. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001, ns, not significant. https://doi.org/10.7554/eLife.44436.009 Murine CypA does not exhibit a dominant negative effect on HCV replication As we observed a lower rescue efficiency of murine compared to human CypA in the Huh7.5-shRNA CypA cells, we considered the possibility that murine CypA may have a dominant negative effect on viral replication versus simply being incompatible. To test this, Huh7.5-shRNA CypA cells were dually transduced with the bicistronic mouse CypA lentivirus expressing eGFP used above and a monocistronic lentivirus containing a C-terminally triple FLAG-tagged hCypA (Figure 4a, Figure 4—figure supplement 1). The expression of the latter, with the expected shift in size, was confirmed via western blot (Supplementary file 1). These dually transduced cells, along with singly transduced controls, were then infected with Jc1-Gluc at an MOI of 0.1 and assessed five dpi by NS5A staining (Figure 4b). We gated on cells that were highly dually positive for both mouse and human CypA and examined in this gate the fraction of HCV NS5A antigen-bearing cells. The presence of elevated human CypA along with murine CypA in the same cells did not result in a significant decline in infection, indicating no dominant negative effect. Figure 4 with 1 supplement see all Download asset Open asset Mouse CypA does not have a dominant negative effect on human CypA in dually transduced cells. Huh7.5-shRNA CypA cells non-transduced or transduced with a 3x-FLAG-tagged human CypA, mouse CypA (expressing eGFP), or both (A) were infected with Jc1-Gluc (MOI = 0.1). (B) At five dpi, cells were stained with antibodies against FLAG and NS5A for flow cytometry analysis. The percentage of NS5A+ cells was determined from the subset of cells that were FLAG+ for the samples singly transduced with the human CypA construct, eGFP +for the samples singly transduced with the mouse CypA construct or FLAG+/eGFP+ for the cells dually transduced with both the human and mouse CypA constructs. In the latter case, cells with high dual transduction were gated on as shown in (A) and the percentage of NS5A + cells determined from this subset. Data shown represent three independent experiments, each performed in triplicate. Two of the data points for the dually transduced, non-infected cells were zero and thus could not be plotted on a log axis. Lines and error bars represent the mean ± SD. Two-way ANOVA with Sidak multiple comparisons test used for statistical analysis. **, p<0.01; ****, p<0.0001; ns, not significant. https://doi.org/10.7554/eLife.44436.011 Triply humanized murine CypA supports HCV spread and release of infectious particles as efficiently as human CypA Although we readily observed replication of the Jc1-Gluc genome in our rescue lines by our luminometry readout, we also tested whether this replication was occurring in only the subset of cells initially infected by the inoculum or spreading across the culture over time (Figure 5a). We took the parental Huh7.5-shRNA CypA cells plus the three rescue lines that displayed replication (mouse, human, or triply humanized mouse CypA) and infected them with Jc1-Gluc at an MOI of 0.1. At three and five dpi, viral spread was assessed by NS5A staining (Figure 5b), which significantly increased over time only for the +human CypA and +triply humanized mouse CypA cultures. Although significantly less compared to these two lines, the number of NS5A positive cells in the mouse rescue line was still significantly higher than that of the non-transduced Huh7.5-shRNA CypA cells. As the NS5A staining is less sensitive compared to the luminometry assay, we wanted to further confirm that infectious particle production was occurring and thus contributing to viral spread. Supernatants from the infected CypA rescue lines were also collected at three and five dpi, applied to naïve Huh7.5 cells and replication assessed three dpi by luminometry (Figure 5c). As expected, the supernatants collected three dpi from all rescue lines resulted in lower replication in Huh7.5 cells compared to the five dpi supernatants, indicating an increase in infectious particle production over time. Supernatants collected from parental Huh7.5 shRNA CypA cells did not exhibit an increase in infectious particles over time, as replication levels in Huh7.5 cells did not significantly increase following infection with the five dpi supernatant. However, there clearly was still some infectious particle production occurring as the level of replication was at least a log above background. Figure 5 Download asset Open asset Viral spread and infectious particle production observed over time in HCV-infected rescue lines. (A) Schematic of experimental workflow. SN, supernatant; Gluc, Gaussia luciferase. Image created with BioRender. (B) Huh7.5-shRNA CypA cells non-transduced or transduced with human, mouse or triply humanized mouse CypA were infected with Jc1-Gluc (MOI = 0.1). At three and five dpi, as represented by circles and squares, respectively, the percentage of NS5A cells compared to naïve cells was assessed by flow cytometry. Note that one human sample had too few cells, so the NS5A staining is shown for only five, instead of six, samples. (C) Supernatants were collected from the infected and naïve cultures at three and five dpi and used to infect naïve Huh7.5 cells. From these infected Huh7.5 cells, supernatants were then collected at three dpi and luciferase activity once more assessed. Circles and squares indicate, respectively, the supernatants collected at 3 and 5 dpi following the infection for which NS5A staining is shown in (B). Results shown are from two representative experiments, each with triplicate samples. Lines and error bars represent the mean ± SD. Ordinary two-way ANOVA test performed followed by Sidak's multiple comparison test, with the mean value for each cell line at three dpi compared to its mean at five dpi. *, p<0.05; ****, p<0.0001, ns, not significant. https://doi.org/10.7554/eLife.44436.013 Characterizing HCV replication in an engineered murine hepatoma line We next moved into a murine context to see how overexpression of murine CypA, human CypA or our triply humanized/murinized mutants might impact HCV replication. To this end, we generated murine Hep56.1D hepatoma cells expressing via lentiviral transduction a variety of factors already established as important to the HCV life cycle: the four HCV human entry factors discussed above (OCLN, CLDN1, SCARBI, and CD81); miR-122 to aid in replication; and SEC14L2, which is absent in hepatoma cells but well-expressed in primary human hepatocytes and has allowed for the in vitro replication and low level viral particle production of normally non-permissive genotypes and clinical isolates of HCV (Saeed et al., 2015). This line, termed Clone 8, we also transduced with murine ApoE (mApoE), which as described above serves in viral packaging and release (Frentzen et al., 2014; Long et al., 2011), to form Clone 8 + ApoE cells (Figure 6a). Expression of all these factors was verified by a combination of flow cytometry, western blot and RT-qPCR (Figure 6b–d) and the replicative kinetics of Jc1-Gluc assessed over six days, with Huh7 cells serving as a positive control (Figure 6e). Replication was consistently highest in the Huh7 cells, with the difference between the Clone 8/Clone 8 + ApoE and Huh7 cells increasing over time till by six dpi there was an approximately three-log difference in luciferase activity. The levels of replication in the parental Hep56
