Stable Clones Research Articles

Abstract 3722: Exploring the role of eIF4E in cancer cells with targeted protein degradation

Abstract Initiation of translation is considered the main rate-limiting step of protein synthesis and requires the recognition of 5’ m7G-cap on mature mRNAs and the formation of eukaryotic translation initiation factor 4F (eIF4F) multi-protein mRNA cap-binding complex. Formation of this complex requires the interaction of eIF4E and the scaffold protein eIF4G, and RNA helicase eIF4A. This eIF4F complex along with eIF3 mediate the recruitment of the 40S ribosomal particle to the 5′ cap of mRNA. Activation of eIF4E is a regulatory hub of many major oncogenic pathways, thus, targeting eIF4E has emerged as a potential therapeutic strategy in cancer. Here we have used a targeted protein degradation approach coupled with genetic rescue to explore the molecular and cellular dependency of cancer cells on eIF4E. Stable H1299 human NSCLC clones expressing FKBP12F36V-tagged eIF4E but lacking endogenous eIF4E were established. Treatment of multiple N- or C-tagged-eIF4E clones with dTAGv-1, an FKBP12F36V selective heterobifunctional molecule that recruits VHL, induced rapid degradation of eIF4E to undetectable levels by 6hr exposure. This also resulted in reduced expression of MCL1, a previously reported biomarker of eIF4E activity. Longer exposures to dTAGv-1 resulted in a cytostasis that was not associated with cell death. A diastereomer negative control of dTAGv-1 that cannot recruit VHL did not elicit loss of eIF4E or the downstream events associated with its loss. Global analysis of protein synthesis initiation by RIBOseq and proteome profiling following dTAGv-1 treatment out to 32hr exposure demonstrated surprisingly few alterations in protein expression despite the significant effect on cancer cell growth. We also expressed wild-type or eIF4E mutants predicted to disrupt key functions and determined their ability to rescue molecular or cellular phenotype associate with eIF4E-loss following dTAGv-1 treatment. Expression of wild-type eIF4E completely rescued cell growth and MCL1 expression. A W56A mutant predicted to disrupt mRNA-cap binding was unable to rescue eIF4E loss. In contrast, expression of W73F or S290A mutants (predicted to disrupt eIF4G binding or exhibit reduced eIF4E activity, respectively) were able to rescue the loss of eIF4E. In summary, our rescue experiments show that mRNA-cap binding by eIF4E is required and that eIF4E:eIF4G interaction in cells may be more complex than predicted. This may also explain the challenges associated with developing selective and cellularly potent inhibitors of the eIF4E:eIF4G interaction and that targeting mRNA-cap binding may be a more effective strategy. We predicted that removing eIF4E would impact on the global synthesis of many proteins. However, our data demonstrate that targeting eIF4E leads to limited effects on protein synthesis that remain sufficient to inhibit cancer cell growth. Further experiments are underway to understand which proteins drive this dependency on eIF4E. Citation Format: Swee Y. Sharp, Marianna Martella, Christopher I. Milton, George Ward, Caroline Richardson, Andrew Woodhead, Paul A. Clarke. Exploring the role of eIF4E in cancer cells with targeted protein degradation. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3722.

Abstract 5870: T-cell factor-4 variants regulate PD-L1 expression in liver cancer cells

Abstract Background: We previously identified 14 isoforms of T-cell factor (TCF)-4, a key transcriptional factor in the Wnt signaling pathway, from human hepatocellular carcinoma (HCC) cell lines (Exp Cell Res 2011). The TCF-4J and K pair has been characterized based on the presence (K) or absence (J) of a conserved SxxSS motif at the head of exon 9. TCF-4J-overexpressing HCC cells (J cells) exhibited high tumorigenic potential in contrast to TCF-4K-overexpressing cells (K cells) (PLoS ONE 2012); however, resistance to anti-cancer agents was more prominent in K cells than J cells. Another analysis demonstrated that loss of exon 4 increased resistance to chemotherapeutic agents and induced epithelial-mesenchymal transition (EMT) (Liver Int 2013). EMT and the expression of programmed cell death 1-ligand 1 (PD-L1) is closely associated with each other, and our preliminary study revealed that EMT was regulated by TCF-4 variants, suggesting possible involvement of the variants in the regulation of PD-L1 expression. In general, PD-L1 is known to be upregulated by hypoxia-inducible factor-1α (HIF-1α) under hypoxic conditions. Therefore, if the TCF-4 variants regulate PD-L1 expression under normoxia, it would be a unique mechanism bridging the Wnt signaling pathway and the tumor immune microenvironment (TIME). Aim: To assess whether or not the TCF-4 variants regulate the expression of PD-L1 in cancer cells under normoxia. Methods: HAK-1A, a well-differentiated HCC cell line (Hepatology 1993), was used in this study. TCF-4K mutants (S269A, S272A, and S273A) were prepared with the conversion of serine (S) in the SxxSS motif to alanine (A) by site-directed mutagenesis. HAK-1A-derived stable clones overexpressing TCF-4J, K, and K-mutants (S269A, S272A, and S273A cells, respectively) were established. Empty vector-transfected mock cells (EV cells) were used as a control. Western blot analysis and real-time RT-PCR were employed to evaluate protein and mRNA expression levels, respectively. Results: J cells and K cells expressed PD-L1 mRNA 2-fold and 4-fold of that in the EV cells, respectively. Of note, the PD-L1 expression mRNA was enhanced up to 11-fold in S269A cells, 11-fold in S272A cells, and 17-fold in S273A cells. The increased mRNA expressions were verified in protein levels. Conclusion: PD-L1 expression was regulated by TCF-4 variants in liver cancer cells under normoxic conditions. The finding suggests that the Wnt signaling pathway fine-tunes the TIME by regulating the expression levels of PD-L1 in a HIF-1α-independent manner. Citation Format: Hironori Koga, Yasuko Imamura, Toshimitsu Tanaka, Hideki Iwamoto, Toru Nakamura, Atsutaka Masuda, Takahiko Sakaue, Hiroyuki Suzuki, Hirohisa Yano, Takumi Kawaguchi. T-cell factor-4 variants regulate PD-L1 expression in liver cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5870.

Involvement of matrix metalloproteinase 1 and urokinase-type plasminogen activator in the PKCα-p38 MAPK pathway-mediated progression of human liver cancer cells.

Our previous studies have shown that the plasminogen activator (PA) and matrix metalloproteinases (MMPs) proteinase systems were highly expressed in highly malignant liver cancer cells and regulated by PKCα. This study investigates whether the PKCα regulation of PA and MMPs systems is conducted through p38 mitogen-activated protein kinase (MAPK) signaling and the pathway is responsible for promoting cell progression. We found that the expressions of p38 MAPK in both highly malignant HA22T/VGH and SK-Hep-1 liver cancer cells were higher than that in other lower malignancy liver cancer cells. Since PKCα activates p38 MAPK in progression of liver cancer, we suspected the PKCα/p38 MAPK signaling pathway to be involved in the regulation of MMPs and PA systems. When SK-Hep-1 cells were treated with SB203580 or DN-p38, only MMP-1 and u-PA mRNA expressions decreased. The p38 MAPK inhibition also decreased the cell migration and invasion. In addition, the mRNA decay assays showed that the higher expressions of MMP-1 and u-PA mRNA in SK-Hep-1 cells were due to the alteration of mRNA stability by p38 MAPK inhibition. Zymography of SK-Hep-1 cells treated with siPKCα vector also showed the decrease of the activity of MMP-1 and u-PA and confirmed changes in mRNA level. Furthermore, only the transfection of MKK6 to the siPKCα-treated SK-Hep-1 stable clone cell restored the attenuation of MMP-1 and u-PA expressions. The treatment of SK-Hep-1 cells with either inhibitor of MMP-1 or u-PA reduced migration, and the reduction was enhanced with both inhibitors. In addition, tumorigenesis was also reduced with both inhibitors. These data suggest a novel finding that MMP-1 and u-PA are critical components in PKCα/MKK6/p38 MAPK signaling pathway which mediates liver cancer cell progression, and that the targeting of both genes may be a viable approach in liver cancer treatment.

A CHO stable pool production platform for rapid clinical development of trimeric SARS-CoV-2 spike subunit vaccine antigens.

Protein expression from stably transfected Chinese hamster ovary (CHO) clones is an established but time-consuming method for manufacturing therapeutic recombinant proteins. The use of faster, alternative approaches, such as non-clonal stable pools, has been restricted due to lower productivity and longstanding regulatory guidelines. Recently, the performance of stable pools has improved dramatically, making them a viable option for quickly producing drug substance for GLP-toxicology and early-phase clinical trials in scenarios such as pandemics that demand rapid production timelines. Compared to stable CHO clones which can take several months to generate and characterize, stable pool development can be completed in only a few weeks. Here, we compared the productivity and product quality of trimeric SARS-CoV-2 spike protein ectodomains produced from stable CHO pools or clones. Using a set of biophysical and biochemical assays we show that product quality is very similar and that CHO pools demonstrate sufficient productivity to generate vaccine candidates for early clinical trials. Based on these data, we propose that regulatory guidelines should be updated to permit production of early clinical trial material from CHO pools to enable more rapid and cost-effective clinical evaluation of potentially life-saving vaccines.

MiR-186 induces tetraploidy in arsenic exposed human keratinocytes

Chronic inorganic arsenic (iAs) exposure in drinking water is a global issue affecting >225 million people. Skin is a major target organ for iAs. miRNA dysregulation and chromosomal instability (CIN) are proposed mechanisms of iAs-induced carcinogenesis. CIN is a cancer hallmark and tetraploid cells can better tolerate increase in chromosome number and aberration, contributing to the evolution of CIN. miR-186 is overexpressed in iAs-induced squamous cell carcinoma relative to iAs-induced hyperkeratosis. Bioinformatic analysis indicated that miR-186 targets mRNAs of important cell cycle regulators including mitotic checkpoint serine/threonine kinase B (BUB1) and cell division cycle 27 (CDC27). We hypothesized that miR-186 overexpression contributes to iAs-induced transformation of keratinocytes by targeting mitotic regulators leading to induction of CIN. Ker-CT cells, a near diploid human keratinocyte cell line, were transduced with miR-186 overexpressing or scrambled control lentivirus. Stable clones were isolated after puromycin selection. Clones transduced with lentivirus expressing either a scrambled control miRNA or miR-186 were maintained with 0 or 100 nM iAs for 4 weeks. Unexposed scrambled control clones were considered as passage matched controls. Chronic iAs exposure increased miR-186 expression in miR-186 clones. miR-186 overexpression significantly reduced CDC27 levels irrespective of iAs exposure. The percentage of tetraploid or aneuploid cells was increased in iAs exposed miR-186 clones. Aneuploidy can arise from a tetraploid intermediate. Suppression of CDC27 by miR-186 may lead to impairment of mitotic checkpoint complex formation and its ability to maintain cell cycle arrest leading to chromosome misalignment. As a result, cells overexpressing miR-186 and chronically exposed to iAs may have incorrect chromosome segregation and CIN. These data suggest that dysregulation of miRNA by iAs mediates tetraploidy, aneuploidy and chromosomal instability contributing to iAs-induced carcinogenesis.

The Use of Phage Antibodies for Microbial Cells Detection (Review)

Phage antibody display technology has revolutionized the field of bacterial immunodetection. This technology allows the expression of an antibody fused to the coat protein of a filamentous bacteriophage. The use of phage display makes it possible to obtain high-affinity antibodies by passing the stage of animal immunization, reducing the time for obtaining stable antibody-producing clones from several months to several weeks, significantly reducing the cost of the process. These advantages make phage antibodies an important tool for bacterial detection. The paper presents a brief description of the technological methods for obtaining phage antibodies to microbial cells. The possibilities and prospects for using phage antibodies as a selective agent in analytical systems, including biosensors, are discussed.

Influence of Canonical and Non-Canonical IFNLR1 Isoform Expression on Interferon Lambda Signaling

Interferon lambdas (IFNLs) are innate immune cytokines that induce antiviral cellular responses by signaling through a heterodimer composed of IL10RB and the interferon lambda receptor 1 (IFNLR1). Multiple IFNLR1 transcriptional variants are expressed in vivo and are predicted to encode distinct protein isoforms whose function is not fully established. IFNLR1 isoform 1 has the highest relative transcriptional expression and encodes the full-length functional form that supports canonical IFNL signaling. IFNLR1 isoforms 2 and 3 have lower relative expression and are predicted to encode signaling-defective proteins. To gain insight into IFNLR1 function and regulation, we explored how altering relative expression of IFNLR1 isoforms influenced the cellular response to IFNLs. To achieve this, we generated and functionally characterized stable HEK293T clones expressing doxycycline-inducible FLAG-tagged IFNLR1 isoforms. Minimal FLAG-IFNLR1 isoform 1 overexpression markedly increased IFNL3-dependent expression of antiviral and pro-inflammatory genes, a phenotype that could not be further augmented by expressing higher levels of FLAG-IFNLR1 isoform 1. Expression of low levels of FLAG-IFNLR1 isoform 2 led to partial induction of antiviral genes, but not pro-inflammatory genes, after IFNL3 treatment, a phenotype that was largely abrogated at higher FLAG-IFNLR1 isoform 2 expression levels. Expression of FLAG-IFNLR1 isoform 3 partially augmented antiviral gene expression after IFNL3 treatment. In addition, FLAG-IFNLR1 isoform 1 significantly reduced cellular sensitivity to the type-I IFN IFNA2 when overexpressed. These results identify a unique influence of canonical and non-canonical IFNLR1 isoforms on mediating the cellular response to interferons and provide insight into possible pathway regulation in vivo.

Optimization of recombinant antibody production based on the vector design and the level of metabolites for generation of Ig- producing stable cell lines

BackgroundThe biopharmaceutical industry is significantly growing worldwide, and the Chinese hamster ovary (CHO) cells are used as a main expression host for the production of recombinant monoclonal antibodies. Various metabolic engineering approaches have been investigated to generate cell lines with improved metabolic characteristics for increasing longevity and mAb production. A novel cell culture method based on the 2-stage selection makes it possible to develop a stable cell line with high-quality mAb production. ResultsWe have constructed several design options of mammalian expression vectors for the high production of recombinant human IgG antibodies. Versions for bipromoter and bicistronic expression plasmids different in promoter orientation and cistron arrangements were generated.The aim of the work presented here was to assess a high-throughput mAb production system that integrates the advantages of high-efficiency cloning and stable cell clones to stage strategy selection reducing the time and effort required to express therapeutic monoclonal mAbs.Development of a stable cell line using bicistronic construct with EMCV IRES-long link gave an advantage in high mAb expression and long-term stability. Two-stage selection strategies allowed the elimination of low-producer clones by using metabolic level intensity to estimate the IgG production in the early steps of selection. The practical application of the new method allows to reduce time and costs during stable cell line development.

Complementary Cell Lines for Protease Gene-Deleted Single-Cycle Adenovirus Vectors.

To increase the safety of adenovirus vector (AdV)-based therapy without reducing its efficacy, a single-cycle adenovirus vector (SC-AdV) with a deletion in the protease gene (PS) was developed in order to be used as a substitute for the replication-competent adenovirus (RC-AdV). Since no infectious viral particles are assembled, there is no risk of viral shedding. The complementary cell lines for this developed AdV proved to be suboptimal for the production of viral particles and require the presence of fetal bovine serum (FBS) to grow. In the current study, we produced both stable pools and clones using adherent and suspension cells expressing the PS gene. The best adherent cell pool can be used in the early stages for the generation of protease-deleted adenovirus, plaque purification, and titration. Using this, we produced over 3400 infectious viral particles per cell. Additionally, the best suspension subclone that was cultured in the absence of FBS yielded over 4000 infectious viral particles per cell. Harvesting time, culture media, and concentration of the inducer for the best suspension subclone were further characterized. With these two types of stable cells (pool and subclone), we successfully improved the titer of protease-deleted adenovirus in adherent and suspension cultures and eliminated the need for FBS during the scale-up production. Eight lots of SC-AdV were produced in the best suspension subclone at a scale of 2 to 8.2 L. The viral and infectious particle titers were influenced by the virus backbone and expressed transgene.

N6-Methyladenosine RNA Modification in Host Cells Regulates Peste des Petits Ruminants Virus Replication.

N6-methyladenosine (m6A) modification is a major RNA epigenetic regulatory mechanism. The dynamics of m6A levels in viral genomic RNA and their mRNAs have been shown to have either pro- or antiviral functions, and therefore, m6A modifications influence virus-host interactions. Currently, no reports are available on the effect of m6A modifications in the genome of Peste des petits ruminants virus (PPRV). In the present study, we took PPRV as a model for nonsegmented negative-sense single-stranded RNA viruses and elucidate the role of m6A modification on viral replication. We detected m6A-modified sites in the mRNA of the virus and host cells, as well as the PPRV RNA genome. Further, it was found that the level of m6A modification in host cells alters the viral gene expression. Knockdown of the METTL3 and FTO genes (encoding the m6A RNA modification writer and eraser proteins, respectively) results in alterations of the levels of m6A RNA modifications in the host cells. Experiments using these genetically modified clones of host cells infected with PPRV revealed that both higher and lower m6A RNA modification in the host cells negatively affect PPRV replication. We found that m6A-modified viral transcripts had better stability and translation efficiency compared to the unmodified mRNA. Altogether, from these data, we conclude that the m6A modification of RNA regulates PPRV replication. These findings contribute toward a way forward for developing novel antiviral strategies against PPRV by modulating the dynamics of host m6A RNA modification. IMPORTANCE Peste des petits ruminants virus (PPRV) causes a severe disease in sheep and goats. PPRV infection is a major problem, causing significant economic losses to small ruminant farmers in regions of endemicity. N6-methyladenosine (m6A) is an important RNA modification involved in various functions, including virus-host interactions. In the present study, we used stable clones of Vero cells, having knocked down the genes encoding proteins involved in dynamic changes of the levels of m6A modification. We also used small-molecule compounds that interfere with m6A methylation. This resulted in a platform of host cells with various degrees of m6A RNA modification. The host cells with these different microenvironments were useful for studying the effect of m6A RNA modification on the expression of viral genes and viral replication. The results pinpoint the level of m6A modifications that facilitate the maximum replication of PPRV. These findings will be useful in increasing the virus titers in cultured cells needed for the economical development of the vaccine. Furthermore, the findings have guiding significance for the development of novel antiviral strategies for limiting PPRV replication in infected animals.

Using a modified piggyBac transposon-combined Cre/loxP system to produce selectable reporter-free transgenic bovine mammary epithelial cells for somatic cell nuclear transfer.

Transposon systems are widely used for genetic engineering in various model organisms. PiggyBac (PB) has recently been confirmed to have highly efficient transposition in the mouse germ line and mammalian cell lines. In this study, we used a modified PB transposon system mediated by PB transposase (PBase) mRNA carrying the human lactoferrin gene driven by bovine β-casein promoter to transfect bovine mammary epithelial cells (BMECs), and the selectable reporter in two stable transgenic BMEC clones was removed using cell-permeant Cre recombinase. These reporter-free transgenic BMECs were used as donor cells for somatic cell nuclear transfer (SCNT) and exhibited a competence of SCNT embryos similar to stable transgenic BMECs and nontransgenic BMECs. The comprehensive information from this study provided a modified approach using an altered PB transposon system mediated by PBase mRNA in vitro and combined with the Cre/loxP system to produce transgenic and selectable reporter-free donor nuclei for SCNT. Consequently, the production of safe bovine mammary bioreactors can be promoted.

A glaucoma-associated OPTN polymorphism, M98K sensitizes retinal cells to endoplasmic reticulum stress and tumour necrosis factor α.

Optineurin/OPTN polymorphism, M98K is associated with normal tension glaucoma in certain populations, and genetic evidence shows its interaction with tumour necrosis factor-alpha (TNFα) polymorphism in causing glaucoma. Endoplasmic reticulum (ER) stress is also associated with glaucoma. We hypothesized that M98K-OPTN may sensitize retinal ganglion cells to various types of stress. To test this hypothesis, stable clones of a retinal cell line, 661W, expressing either wild-type (WT)-OPTN or M98K-OPTN were generated and examined for their survival under various stress conditions. Compared with WT-OPTN expressing cells, M98K-OPTN expressing cells showed significantly lower cell survival and higher activation of caspase-3 and caspase-8 upon treatment with tunicamycin (an inducer of ER stress) or TNFα. Levels of ER stress sensors IRE1α, PERK and ATF6 were significantly higher in M98K-OPTN expressing cells. Tunicamycin treatment resulted in significantly higher induction of ER stress marker CHOP and several other ER stress response genes regulated by IRE1α-XBP1, PERK-ATF4 and ATF6 pathways, in M98K-OPTN expressing cells. Splicing of XBP1 and ATF6 activation was higher in tunicamycin-treated M98K-OPTN expressing cells. Increased levels of PERK and IRE1α proteins in M98K-OPTN expressing cells were dependent on autophagy. Overall, our results show that M98K-OPTN sensitizes retinal cells to TNFα and ER stress-induced cell death. We also show that M98K-OPTN alters ER stress response signalling, which possibly enhances the sensitivity of retinal cells to ER stress. Our results provide support to the hypothesis that M98K-OPTN may cooperate with other genetic or environmental factors to cause retinal ganglion cell death associated with glaucoma.

Shortening the biologics clinical timeline with a novel method for generating stable, high-producing cell pools and clones.

Reducing drug development timelines is an industry-wide goal to bring medicines to patients in need more quickly. This was exemplified in the coronavirus disease 2019 pandemic where reducing development timelines had a direct impact on the number of lives lost to the disease. The use of drug substances produced using cell pools, as opposed to clones, has the potential to shorten development timelines. Toward this goal, we have developed a novel technology, GPEx® Lightning, that allows for rapid, reproducible, targeted recombination of transgenes into more than 200 Dock sites in the Chinese hamster ovary cell linegenome. This allows for rapid production of high-expressing stable cell pools and clones that reach titers of 4-12 g/l in generic fed-batch production. These pools and clones are highly stable in both titer and glycosylation, showing strong similarities in glycosylation profiles.

Accidental acquisition of a rescued Japanese encephalitis virus with unspliced introns in the viral genome when using an intron-based stabilization approach

The intron-based stabilization approach is a very useful strategy for construction of stable flavivirus infectious clones. SA14-14-2 is a highly attenuated Japanese encephalitis (JE) live vaccine strain that has been widely used in China since 1989. To develop safe and effective recombinant vaccines with SA14-14-2 as a backbone vector, we constructed the DNA-based infectious clone pCMW-JEV of SA14-14-2 using the intron-based stabilization approach and acquired the rescued virus rDJEV, which retained the biological properties of the parental virus. Unexpectedly, a rescued virus strain with altered virulence, designated rHV-DJEV, was accidentally acquired in one of the transfection experiments. rHV-DJEV showed up to 105-fold increased neurovirulence compared with the SA14-14-2 parental strain. Genome sequencing showed that the inserted introns were still present in the genome of rHV-DJEV. Therefore, we think that the intron-based stabilization approach should be used with caution in vaccine development and direct iDNA immunization.

The development and characterization of a stable Coxsackievirus A16 infectious clone with Nanoluc reporter gene.

Coxsackievirus A16 (CA16) belongs to the Human Enterovirus A species, which is a common pathogen causing hand, foot, and mouth disease in children. Currently, specific vaccines and drugs against CA16 are unavailable, and there is an unmet need to further understand the virus and invent effective treatment. Constructing a CA16 infectious clone with a reporter gene will greatly facilitate its virological studies. Here, we first reported the construction of a CA16 infectious clone (rCA16) whose progeny is highly replicative and virulent in suckling mice. On the basis of rCA16, we further inserted a NanoLuc (Nluc) reporter gene and made the rCA16-Nluc clone. We found that the Nluc gene in rCA16-Nluc is stable during continuous growing in Vero cells and thus allowed detection of a steady luciferase signal in rCA16-Nluc-infected Vero cells over 10 passages. Its application in antivirals characterization and high-throughput screening is exemplified by measuring IC50, CC50, and selection index of guanidine hydrochloride, ribavirin, chloroquine, and ammonium chloride against CA16. Finally, we showed that rCA16-Nluc based assay greatly simplified the CA16 neutralizing antibody tests. Thus, these two CA16 infectious clones will be robust tools for future enterovirus studies and antivirals development.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

The eighth International Forum on Stem Cells: virtual meeting, October 20-21, 2022.

Comparison of busulfan and total body irradiation conditioning on hematopoietic clonal dynamics following lentiviral gene transfer in rhesus macaques

The clonal dynamics following hematopoietic stem progenitor cell (HSPC) transplantation with busulfan conditioning are of great interest to the development of HSPC gene therapies. Compared with total body irradiation (TBI), busulfan is less toxic and more clinically relevant. We used a genetic barcoded HSPC autologous transplantation model to investigate the impact of busulfan conditioning on hematopoietic reconstitution in rhesus macaques. Two animals received lower busulfan dose and demonstrated lower vector marking levels compared with the third animal given a higher busulfan dose, despite similar busulfan pharmacokinetic analysis. We observed uni-lineage clonal engraftment at 1month post-transplant, replaced by multilineage clones by 2 to 3months in all animals. The initial multilineage clones in the first two animals were replaced by a second multilineage wave at 9months; this clonal pattern disappeared at 13months in the first animal, though was maintained in the second animal. The third animal maintained stable multilineage clones from 3months to the most recent time point. In addition, busulfan animals exhibit more rapid HSPC clonal mixing across bone marrow sites and less CD16+ NK-biased clonal expansion compared with TBI animals. Therefore, busulfan conditioning regimens can variably impact the marrow niche, resulting in differences in clonal patterns with implications for HSPC gene therapies.

The Growth Kinetics of Clonal Hematopoiesis Inform Risk of Blood Cancer Progression

Introduction: Clonal hematopoiesis of indeterminate potential (CHIP) is a frequent consequence of aging associated with increased risk of blood cancers, although overall individual risk is < 1% per annum. Proposed risk factors for blood cancer progression include a variant allele frequency (VAF) > 10%, specific gene mutations or the presence of more than one CHIP clone. However, the relative value of monitoring CHIP clone growth with respect to blood cancer risk is unknown. To address this evidence gap, we measured CHIP at baseline and 3 years later in 11,298 healthy individuals aged ≥70y from the ASPREE randomized clinical trial (www.aspree.org). Methods: A custom, high sensitivity (0.5%) and reproducible amplicon-based targeted sequencing assay was designed to assess CHIP in 19 genes, except for SRSF2, which had poor coverage. CHIP was defined as a VAF ≥ 2% with a minimum of 5 variant reads for previously reported CHIP or 20 for variants not previously reported. The analysis involved measuring CHIP variants in two serial blood samples collected at baseline and year 3 of follow-up for longitudinal analysis. Associations between CHIP and blood cancers were assessed using Cox proportional hazards models. Results: CHIP at baseline was present in 34% of participants who had a corresponding year 3 sample with adequate sequencing depth. Unexpectedly, 1,245/3,110 (40%) CHIP clones present at baseline fell below 2%, while 1,520 new CHIP clones appeared at 3 years. This equated to an incident CHIP rate of 7% per annum. CHIP genes more likely to expand and contract were TP53 and ASXL1 (odds ratio 1.25 and 1.22 respectively). To measure growth characteristics of CHIP clones, we calculated a Fitness Score, FS (Log2(VAF3-year/VAFBaseline) for the 3,110 clones present at baseline. We observed no significant mean fitness advantage for any recurrent CHIP variant other than PPM1D R552* (Figure 1). CHIP variants of DNMT3A (including DNMT3A R882), TET2 and ASXL1 had a statistically significant negative FS. Even variants generally considered most pathogenic (SF3B1 K700E, JAK2 V617F, U2AF1 Q157, TP53 R273) had no significant fitness advantage. Some CHIP clones had a significant growth disadvantage (FS < -1). For example, only 1/28 DNMT3A S393N clones present at baseline remained above 2% and almost half of TP53 variants reduced in size by more than 2-fold at 3 years. We observed 299 blood cancers over a median time of 6.9 years. Individuals with CHIP clones >10% VAF (n= 652) had increased incidence of blood cancers (adjusted hazard ratio, aHR 2.14, confidence interval, CI 1.34-3.42, p=0.002). Genes associated with increased risk for blood cancers were SF3B1/U2AF1 (aHR 3.02, CI 1.63-5.61, p<0.001) and TET2 (n=aHR 1.89, CI 1.18-3.02, p=0.008). Participants with small clones (VAF 2-10%) at baseline or year 3 had no increased risk for blood cancers compared to those without CHIP. To investigate if the growth kinetics of CHIP predicts blood cancer risk, we subdivided CHIP clones into expanding (FS > +1, n= 104), stable (FS -1 to +1, n= 2043) and contracting (FS < -1, n= 963). Baseline VAF of expanding clones were smaller than stable clones: geometric mean 3.9% and 5.0%, respectively (p<0.001). Expanding clones were enriched 2-fold for PPM1D, SF3B1 and JAK2 V617F. Participants with expanding clones (n=99) were more likely to have more than one CHIP variant (50% compared with 39% for stable and 37% for contracting), although 90% of these additional variants were stable or contracting, indicating that the expanding clone was dominant. For similar VAF levels at 3 years, participants with expanding CHIP clones had a higher risk of blood cancer than a stable or contracting one (Figure 2). Conclusions: This is the largest longitudinal study of CHIP variants and associated blood cancer outcomes to date. In contrast to other longitudinal studies, there was little evidence for selective growth advantage of CHIP clones with a VAF ≥ 2%, including those with pathogenic mutations of DNMT3A, SF3B1, U2AF1, TP53 and JAK2. While clone size (VAF > 10%) and gene mutation (SF3B1, U2AF1 and TET2) were associated with increased risk of blood cancer progression, our study cautions clinical intervention of CHIP based upon a single timepoint analysis irrespective of the gene mutation. Instead, we propose monitoring of clone size may be a clinically more useful strategy to inform counselling and patient selection for early intervention studies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Mechanism of CD79A and CD79B Support for IgM+ B Cell Fitness through B Cell Receptor Surface Expression.

The BCR consists of surface-bound Ig and a heterodimeric signaling unit comprised of CD79A and CD79B. Upon cognate Ag recognition, the receptor initiates important signals for B cell development and function. The receptor also conveys Ag-independent survival signals termed tonic signaling. Although the requirement of a CD79A/CD79B heterodimer for BCR complex assembly and surface expression is well established based on mice models, few studies have investigated this in human mature B cells. In this study, we found that human tonsillar B cells with high surface expression of IgM or IgG had potentiated BCR signaling compared with BCRlow cells, and high IgM expression in germinal center B cells was associated with reduced apoptosis. We explored the mechanism for IgM surface expression by CRISPR/Cas9-induced deletion of CD79A or CD79B in four B lymphoma cell lines. Deletion of either CD79 protein caused loss of surface IgM in all cell lines and reduced fitness in three. From two cell lines, we generated stable CD79A or CD79B knockout clones and demonstrated that loss of CD79A or CD79B caused a block in N-glycan maturation and accumulation of immature proteins, compatible with retention of BCR components in the endoplasmic reticulum. Rescue experiments with CD79B wild-type restored surface expression of CD79A and IgM with mature glycosylation, whereas a naturally occurring CD79B G137S mutant disrupting CD79A/CD79B heterodimerization did not. Our study highlights that CD79A and CD79B are required for surface IgM expression in human B cells and illuminates the importance of the IgM expression level for signaling and fitness.

Long-Term Clonal Inversion in an MDS-RS Case with Dual SF3B1 Mutations

Recent large-scale studies have traced SF3B1 mutations as a driving event in clonal establishment and expansion, and a hallmark of myelodysplastic syndrome with ring sideroblasts (MDS-RS). However, the mechanisms underlying the fitness advantage allowing SF3B1-mutant (SF3B1mt) clones to thrive remain poorly understood. Thus, tracking these events in conditions of low noise is crucial to our comprehension of clonal hematopoiesis. Here we report a 12-year clinical and molecular follow-up of a unique dual SF3B1mt MDS-RS case displaying a dynamic state of clonal competition between two clones propagated by a N626D and K666N mutation, respectively (Panel A). After an initial rapid expansion of the N626D clone compared to the relatively stable K666N clone during the initial follow-up period of ~2 years, the K666N clone spontaneously expanded at expense of the N626D clone to eventually dominate the bone marrow, driving a complete inversion of clonal prevalence. Clinically, these active shifts of high clonal involvement in the bone marrow were accompanied by stable subclinical anemia (median 12.5 g/L, range 12.0 - 13.2 g/L) and a continued decrease in the ring sideroblast phenotype over time (from 42% to 8% RS) in the absence of any treatment. The stability of this patient's clinical profile was therefore considered as unlikely to confound further characterization. Through a combination of whole-genome sequencing (WGS) of hematopoietic stem and progenitor cell (HSPC)-generated colony-forming units (CFU), combined DNA + RNAseq (TargetSeq) of FACS-purified hematopoietic stem cells (HSCs) and megakaryocyte-erythroid progenitor cells (MEP), and high-throughput 3'-based 10X Genomics single-cell RNAseq of CD34+ HSPCs, we molecularly characterized the two distinct SF3B1mt clones in bone marrow samples acquired before, during and after the points of clonal inversion (Panel B). Using WGS of single cell-derived colonies, we established that the acquisition of either the N626D or K666N mutation had preceded the diagnosis of MDS-RS by at least 20 years. Moreover, both mutations were associated with an increased mutational rate as compared to wildtype colonies, and the two clones presented distinct patterns of additional coding mutations as potential co-drivers of expansion. Mirroring previous characterizations of SF3B1mt HSPCs, we identified a common and deleterious alternative splicing profile to both genotypes via TargetSeq which included mis-splicing events in the known SF3B1mt targets MAP3K7 and SEPTIN6. In addition, we identified mutation-specific splicing profiles as potentially responsible for distinct downstream distinctions between both clones. Through this analysis, we identified the K666N-specific loss of expression of the tumor suppressors HOXA5, RGCC and NEURL1 as potential compound factors to the known higher risk underlying SF3B1K666N mutations. Finally, we disentangled clone-driven and aging-driven transcriptomic changes to identify a significant decrease of the early hematopoietic controllers ID1, ID2 and ID3 as a potential mechanism of aging-related cellular senescence. In conclusion, this case study comprises a unique long-term follow-up of the clonal dynamics underlying two co-existing, distinct and competing SF3B1mt clones which identifies subtle molecular changes and differences underlying the expansion and progression of SF3B1mt clones. *Moura PL, Hofman IJF, Nannya Y and Aliouat A contributed equally to this work. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal