Single Amino Acid Substitution Research Articles

The interaction of GRP78 and Zika virus E and NS1 proteins occurs in a chaperone-client manner

Glucose regulated protein 78 (GRP78) is a chaperone protein that is a central mediator of the unfolded protein response, a key cellular stress response pathway. GRP78 has been shown to be critically required for infection and replication of a number of flaviviruses, and to interact with both non-structural (NS) and structural flavivirus proteins. However, the nature of the specific interaction between GRP78 and viral proteins remains largely unknown. This study aimed to characterize the binding domain and critical amino acid residues that mediate the interaction of GRP78 to ZIKV E and NS1 proteins. Recombinant EGFP fused GRP78 and individual subdomains (the nucleotide binding domain (NBD) and the substrate binding domain (SBD)) were used as a bait protein and co-expressed with full length or truncated ZIKV E and NS1 proteins in HEK293T/17 cells. Protein–protein interactions were determined by a co-immunoprecipitation assay. From the results, both the NBD and the SBD of GRP78 were crucial for an effective interaction. Single amino acid substitutions in the SBD showed that R492E and T518A mutants significantly reduced the binding affinity of GRP78 to ZIKV E and NS1 proteins. Notably, the interaction of GRP78 with ZIKV E was stably maintained against various single amino acid substitutions on ZIKV E domain III and with all truncated ZIKV E and NS1 proteins. Collectively, the results suggest that the principal binding between GRP78 and viral proteins is mainly a classic canonical chaperone protein-client interaction. The blocking of GRP78 chaperone function effectively inhibited ZIKV infection and replication in neuronal progenitor cells. Our findings reveal that GRP78 is a potential host target for anti-ZIKV therapeutics.

Single Amino Acid Substitution in the Matrix Protein of Rabies Virus Is Associated with Neurovirulence in Mice

Rabies is a fatal encephalitic infectious disease caused by the rabies virus (RABV). RABV is highly neurotropic and replicates in neuronal cell lines in vitro. The RABV fixed strain, HEP-Flury, was produced via passaging in primary chicken embryonic fibroblast cells. HEP-Flury showed rapid adaptation when propagated in mouse neuroblastoma (MNA) cells. In this study, we compared the growth of our previously constructed recombinant HEP (rHEP) strain—based on the sequence of the HEP (HEP-Flury) strain—with that of the original HEP strain. The original HEP strain exhibited higher titer than rHEP and a single substitution at position 80 in the matrix (M) protein M(D80N) after incubation in MNA cells, which was absent in rHEP. In vivo, intracerebral inoculation of the rHEP-M(D80N) strain with this substitution resulted in enhanced viral growth in the mouse brain and a significant loss of body weight in the adult mice. The number of viral antigen-positive cells in the brains of adult mice inoculated with the rHEP-M(D80N) strain was significantly higher than that with the rHEP strain at 5 days post-inoculation. Our findings demonstrate that a single amino acid substitution in the M protein M(D80N) is associated with neurovirulence in mice owing to adaptation to mouse neuronal cells.

Mutations in the NUP93, NUP107 and NUP160 genes cause steroid-resistant nephrotic syndrome in Chinese children

BackgroundThe variants of nucleoporins are extremely rare in hereditary steroid-resistant nephrotic syndrome (SRNS). Most of the patients carrying such variants progress to end stage kidney disease (ESKD) in their childhood. More clinical and genetic data from these patients are needed to characterize their genotype–phenotype relationships and elucidate the role of nucleoporins in SRNS.MethodsFour patients of SRNS carrying biallelic variants in the NUP93, NUP107 and NUP160 genes were presented. The clinical and molecular genetic characteristics of these patients were summarized, and relevant literature was reviewed.ResultsAll four patients in this study were female and initially presented with SRNS. The median age at the onset of the disease was 5.08 years, ranging from 1 to 10.5 years. Among the four patients, three progressed to ESKD at a median age of 7 years, ranging from 1.5 to 10.5 years, while one patient reached stage 3 chronic kidney disease (CKD3). Kidney biopsies revealed focal segmental glomerulosclerosis in three patients. Biallelic variants were detected in NUP93 in one patient, NUP107 in two patients, as well as NUP160 in one patient respectively. Among these variants, five yielded single amino acid substitutions, one led to nonsense mutation causing premature termination of NUP107 translation, one caused a single nucleotide deletion resulting in frameshift and truncation of NUP107. Furthermore, one splicing donor mutation was observed in NUP160. None of these variants had been reported previously.ConclusionThis report indicates that biallelic variants in NUP93, NUP107 and NUP160 can cause severe early-onset SRNS, which rapidly progresses to ESKD. Moreover, these findings expand the spectrum of phenotypes and genotypes and highlight the importance of next-generation sequencing in elucidating the molecular basis of SRNS and allowing rational treatment for affected individuals.

[A pedigree of myotonia congenita with a novel mutation p.F343C of the CLCN1 gene].

A Japanese woman experienced slowness of movement in her early teens and difficulty in opening her hands during pregnancy. On admission to our hospital at 42 years of age, she showed grip myotonia with warm-up phenomenon. However, she had neither muscle weakness, muscle atrophy, cold-induced symptomatic worsening nor episodes of transient weakness of the extremities. Needle electromyography of the first dorsal interosseous and anterior tibial muscles demonstrated myotonic discharges. Whole exome sequencing of the patient revealed a heterozygous single-base substitution in the CLCN1 gene (c.1028T>G, p.F343C). The same substitution was identified in affected members of her family (mother and brother) by Sanger sequencing, but not in healthy family members (father and a different brother). We diagnosed myotonia congenita (Thomsen disease) with a novel CLCN1 mutation in this pedigree. This mutation causes a single amino acid substitution in the I-J extracellular loop region of CLCN1. Amino acid changes in the I-J loop region are rare in an autosomal-dominantly inherited form of myotonia congenita. We think that this pedigree is precious to understand the pathogenesis of myotonia congenita.

Variant Effect Prediction in the Age of Machine Learning.

Over the years, many computational methods have been created for the analysis of the impact of single amino acid substitutions resulting from single-nucleotide variants in genome coding regions. Historically, all methods have been supervised and thus limited by the inadequate sizes of experimentally curated data sets and by the lack of a standardized definition of variant effect. The emergence of unsupervised, deep learning (DL)-based methods raised an important question: Can machines learn the language of life from the unannotated protein sequence data well enough to identify significant errors in the protein "sentences"? Our analysis suggests that some unsupervised methods perform as well or better than existing supervised methods. Unsupervised methods are also faster and can, thus, be useful in large-scale variant evaluations. For all other methods, however, their performance varies by both evaluation metrics and by the type of variant effect being predicted. We also note that the evaluation of method performance is still lacking on less-studied, nonhuman proteins where unsupervised methods hold the most promise.

Non-Invasive Delivery of Negatively Charged Nanobodies by Anodal Iontophoresis: When Electroosmosis Dominates Electromigration.

Iontophoresis enables the non-invasive transdermal delivery of moderately-sized proteins and the needle-free cutaneous delivery of antibodies. However, simple descriptors of protein characteristics cannot accurately predict the feasibility of iontophoretic transport. This study investigated the cathodal and anodal iontophoretic transport of the negatively charged M7D12H nanobody and a series of negatively charged variants with single amino acid substitutions. Surprisingly, M7D12H and its variants were only delivered transdermally by anodal iontophoresis. In contrast, transdermal permeation after cathodal iontophoresis and passive diffusion was <LOQ. The anodal iontophoretic delivery of these negatively charged proteins was achieved because electroosmosis was the dominant electrotransport mechanism. Cutaneous deposition after the anodal iontophoresis of M7D12HWT (wild type), and the R54E and K65E variants, was statistically superior to that after cathodal iontophoresis (6.07 ± 2.11, 9.22 ± 0.80, and 14.45 ± 3.45 μg/cm2, versus 1.12 ± 0.30, 0.72 ± 0.27, and 0.46 ± 0.07 µg/cm2, respectively). This was not the case for S102E, where cutaneous deposition after anodal and cathodal iontophoresis was 11.89 ± 0.87 and 8.33 ± 2.62 µg/cm2, respectively; thus, a single amino acid substitution appeared to be sufficient to impact the iontophoretic transport of a 17.5 kDa protein. Visualization studies using immunofluorescent labeling showed that skin transport of M7D12HWT was achieved via the intercellular and follicular routes.

Identification of Critical Amino Acid Residues of a Two-Component Sensor Protein for Signal Sensing in Porphyromonas gingivalis Fimbriation via Random Mutant Library Construction.

Porphyromonas gingivalis (Pg) utilizes FimA fimbriae to colonize the gingival sulcus and evade the host immune system. The biogenesis of all FimA-related components is positively regulated by the FimS-FimR two-component system, making the FimS sensory protein an attractive target for preventing Pg infection. However, the specific environmental signal received by FimS remains unknown. We constructed random Pg mutant libraries to identify critical amino acid residues for signal sensing by FimS. Optimized error-prone polymerase chain reaction (PCR) was used to introduce a limited number of random mutations in the periplasmic-domain-coding sequence of fimS, and expression vectors carrying various mutants were generated by inverse PCR. More than 500 transformants were obtained from the fimS-knockout Pg strain using the Escherichia coli-Pg conjugal transfer system, whereas only ~100 transformants were obtained using electroporation. Four and six transformant strains showed increased and decreased fimA expression, respectively. Six strains had single amino acid substitutions in the periplasmic domain, indicating critical residues for signal sensing by FimS. This newly developed strategy should be generally applicable and contribute to molecular genetics studies of Pg, including the elucidation of structure-function relationships of proteins of interest.

6Pgdh polymorphism in wild bulb mite populations: prevalence, environmental correlates and life history trade-offs.

Genetic polymorphism in key metabolic genes plays a pivotal role in shaping phenotypes and adapting to varying environments. Polymorphism in the metabolic gene 6-phosphogluconate dehydrogenase (6Pgdh) in bulb mites, Rhizoglyphus robini is characterized by two alleles, S and F, that differ by a single amino acid substitution and correlate with male reproductive fitness. The S-bearing males demonstrate a reproductive advantage. Although the S allele rapidly fixes in laboratory settings, the persistence of polymorphic populations in the wild is noteworthy. This study examines the prevalence and stability of 6Pgdh polymorphism in natural populations across Poland, investigating potential environmental influences and seasonal variations. We found widespread 6Pgdh polymorphism in natural populations, with allele frequencies varying across locations and sampling dates but without clear geographical or seasonal clines. This widespread polymorphism and spatio-temporal variability may be attributed to population demography and gene flow between local populations. We found some correlation between soil properties, particularly cation content (Na, K, Ca, and Mg) and 6Pgdh allele frequencies, showcasing the connection between mite physiology and soil characteristics and highlighting the presence of environment-dependent balancing selection. We conducted experimental fitness assays to determine whether the allele providing the advantage in male-male competition has antagonistic effects on life-history traits and if these effects are temperature-dependent. We found that temperature does not differentially influence development time or juvenile survival in different 6Pgdh genotypes. This study reveals the relationship between genetic variation, environmental factors, and reproductive fitness in natural bulb mite populations, shedding light on the dynamic mechanisms governing 6Pgdh polymorphism.

Deep mutational scanning of the RNase III-like domain in Trypanosoma brucei RNA editing protein KREPB4.

Kinetoplastid pathogens including Trypanosoma brucei, T. cruzi, and Leishmania species, are early diverged, eukaryotic, unicellular parasites. Functional understanding of many proteins from these pathogens has been hampered by limited sequence homology to proteins from other model organisms. Here we describe the development of a high-throughput deep mutational scanning approach in T. brucei that facilitates rapid and unbiased assessment of the impacts of many possible amino acid substitutions within a protein on cell fitness, as measured by relative cell growth. The approach leverages several molecular technologies: cells with conditional expression of a wild-type gene of interest and constitutive expression of a library of mutant variants, degron-controlled stabilization of I-SceI meganuclease to mediate highly efficient transfection of a mutant allele library, and a high-throughput sequencing readout for cell growth upon conditional knockdown of wild-type gene expression and exclusive expression of mutant variants. Using this method, we queried the effects of amino acid substitutions in the apparently non-catalytic RNase III-like domain of KREPB4 (B4), which is an essential component of the RNA Editing Catalytic Complexes (RECCs) that carry out mitochondrial RNA editing in T. brucei. We measured the impacts of thousands of B4 variants on bloodstream form cell growth and validated the most deleterious variants containing single amino acid substitutions. Crucially, there was no correlation between phenotypes and amino acid conservation, demonstrating the greater power of this method over traditional sequence homology searching to identify functional residues. The bloodstream form cell growth phenotypes were combined with structural modeling, RECC protein proximity data, and analysis of selected substitutions in procyclic form T. brucei. These analyses revealed that the B4 RNaseIII-like domain is essential for maintenance of RECC integrity and RECC protein abundances and is also involved in changes in RECCs that occur between bloodstream and procyclic form life cycle stages.

Abstract SY16-01: What IDH mouse models have taught us about hematological diseases, from leukemia to T cell lymphoma

Abstract More than fifteen years after their discovery, IDH mutations remain as intriguing as ever. Three mutational hotspots are described in different cancers, including glioma, chondrosarcoma, or hematological diseases, resulting in single amino acid substitutions in two of the IDH genes (IDH1R132, IDH2R172 and IDH2R140). Wild-type IDH1/2 catalyze the conversion of isocitrate into α-ketoglutarate (α-KG), and their mutations confer neomorphic activity to this metabolic enzyme, reducing α-KG to 2-hydroxyglutarate (2-HG). 2-HG is a potent inhibitor of several dioxygenases that normally use α-KG as a cofactor. Among them is the ten-eleven translocation (TET) family, which catalyzes the hydroxylation of methylated cytosine bases in the DNA. Mutations in epigenetic regulators, such as IDH, are commonly found in hematological malignancies including myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and peripheral T cell lymphoma (PTCL), and especially angioimmunoblastic T cell lymphoma (AITL). Despite many advances, these hematologic diseases still have significant unmet medical needs, and require novel approaches to patient selection and therapy. IDH inhibitors are currently under investigation in MDS, AML and PTCL and have shown promise in early clinical trials for AML and MDS. However, not all IDH-mutant patients respond, and resistance frequently develops. Changes to epigenetic regulation and diversity driven by IDH1/2 mutations likely contribute to these effects, but our incomplete understanding of the underlying biology limits therapeutic intervention. Establishing the relationship between tumor genetics and human diseases in the context of IDH mutations is challenging. The distribution of the three hotspot mutations among the different pathologies is complex and not yet fully understood. An intriguing fact is that IDH2R140 mutation is exclusively found in myeloid malignancies. Furthermore, clinical data indicate that the co-mutational pattern of IDH1/2 mutations is associated differently in hematological disorders. For instance, IDH1/2 and TET2 mutations are essentially mutually exclusive in myeloid context but frequently co-occur in AITL, suggesting that their effects in specific cell types and disease contexts may differ. Understanding how IDH1/2 and TET2 mutations drive malignancy and why they cooperate differently in myeloid and lymphoid diseases can help distinguish common from specific mechanisms in myeloid and lymphoid compartments, revealing specific tumor cell vulnerabilities, which could be targeted to improve patient outcomes. The laboratory of Professor Mak has generated three strains of Idh knock-in mice, each bearing one of the three hotspot mutations and has assembled a multidisciplinary team in epigenetics and clinical investigation to explore their role and set the stage for further mechanistic insight into mutant IDH, both in myeloid or lymphoid contexts. 2-HG levels differ between the myeloid and lymphoid lineages of each strain and may explain the variations in the mutational spectrum associated with different cellular contexts. Even within the myeloid lineage, analyses of these three Idh-mutant mice suggest that these alleles produce phenotypes of varying severity, correlating with 2-HG levels, with Idh2R172 exhibiting the most severe phenotype. We showed that altered DNA and histone methylation; partially block hematopoietic cell differentiation at the hematopoietic stem cell/progenitor stage; resulting in a hematopoietic phenotype reminiscent of human MDS. Given the mutual exclusivity of IDH1/2 and TET2 mutations in myeloid neoplasms, it has been hypothesized that they converge on a common oncogenic pathway. However, in a first study, we demonstrated that mutant Idh1 impairs DNA repair via a mechanism independent of Tet2 at the stem cell stage. Then, in a second study, we showed that these alterations have distinct, and even opposite, effects on hematopoietic stem cells and progenitors. Epigenetic and single-cell transcriptomic analyses revealed that Idh2R172 and Tet2 mutations had diverging consequences. Our data argue against a simple equivalence of Idh1/2 and Tet2 mutations, and may help to explain some of the clinical differences between TET2- and IDH-mutant in a myeloid context. In PTCL, the neoplastic cells represent a minor part of the tumor, a truth that is even more pronounced in AITL, where the microenvironment frequently accounts for 90% of the tumoral mass. Using molecular and histological approaches, it has been shown that driving mutations, such as TET2, occur in hematopoietic stem cells, and are found in both malignant and non-malignant cells. On the other hand, others alterations, such as IDH2, occur in mature T cells, specifically in T follicular helper (TFH) cells, and are restrictive to malignant cells. Our team, in collaboration with a group of researchers led by Drs. Philippe Gaulard and François Lemonnier, demonstrated that TET and IDH mutations co-occur in TFH malignant cells. We showed increased levels of 2-HG both in tissue samples and serum of IDH2R172-mutated AITL patients, although these levels are lower than in AML patients. This difference could either be attributed to a lower tumor burden in AITL than in AML, or to the difference of the mutant enzyme ability to produce 2-HG based on the cellular context (myeloid cells versus lymphoid cells). Notably, IDH2 mutation without TET2 mutation is really rare in AITL. However, the reason why IDH2 mutation drives oncogenesis only in the presence of TET2 mutation is still not fully defined. After generating the first AITL mouse model mutated for Idh2 and Tet2, we demonstrated that the double mutant T cells produce more 2-HG than the single Idh2-mutated T cells, potentially resulting in a greater inhibition of αKG-dependent dioxygenases. This heightened ability to synthesize 2-HG could be linked to the altered metabolic gene expression programs in Tet2-mutated Tfh cells, although this hypothesis requires further confirmation. Ultimately, the combination of Idh2 and Tet2 mutations in TFH cells driving this disease undergoes extensive genetic and epigenetic reprogramming, mainly affecting their interactions with nearby cells in the microenvironment. Particularly, these mutations profoundly impact the crosstalk between neoplastic Tfh cells and the surrounding B cells, leading to the expansion of B and plasma cells in these mice, sometimes clonally. This recently discovered crosstalk among various immune cell types within the tumor microenvironment highlights the need to define how this cooperation occurs and what might disrupt its occurrence to forestall disease progression. Understanding the involvement of immune cells in the microenvironment in hematopoietic diseases holds the potential for significant advances in their treatment. In conclusion, genetically engineered mouse models serve as powerful tools to comprehend the implications of IDH mutations in myeloid or T cell biology. Our research shed new light on the mechanisms by which mutations in Idh1/2 and Tet2 drive the development and progression of leukemias and lymphomas. Through a multifaceted approach conducted in our genetically engineered mouse models, we have identified mutation-specific and disease-specific effects. This innovative approach has led to significant basic research discoveries which can now be evaluated in patient samples. These findings may help to select patients and devise additional treatment strategies using existing and investigational therapies, which can then be translated to preclinical testing. Citation Format: Julie Leca. What IDH mouse models have taught us about hematological diseases, from leukemia to T cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr SY16-01.

Abstract NG05: Epitope editing enables targeted immunotherapy of acute myeloid leukemia

Abstract Introduction. Chimeric antigen receptor T cells (CAR-T), bispecific and antibody-drug conjugates are promising adoptive immunotherapies that can overcome the limitations of conventional cancer treatments and have demonstrated striking efficacy when targeting dispensable lineage antigens (Ag), e.g. CD19 for B-ALL. Nonetheless, the absence of safely actionable tumor-restricted markers hampers their application to other hematological malignancies, such as acute myeloid leukemia (AML). Since AML shares most surface markers with normal hematopoietic stem/progenitor cells (HSPC) or differentiated myeloid cells, on-target/off-tumor toxicities would result in myeloid aplasia and impairment of hematopoietic reconstitution. Furthermore, due to AML intra-tumoral heterogeneity, targeting more than one Ag may be required, exacerbating the risk of overlapping toxicity. Despite this, AML immunotherapies are currently under development, but their role will likely be restricted to bridge treatment before allogeneic HSPC transplantation (HSCT), decreasing the chances of AML eradication. Removal of targeted Ag through CRISPR-Cas KO from donor HSPCs used in HSCT has recently been proposed, but this can only be applied to genes dispensable for hematopoietic function. However, targeting irrelevant genes may facilitate tumor escape through Ag downregulation. Here, we show that precise editing of the targeted epitope within FLT3, KIT (CD117) and IL3RA (CD123) in HSPCs results in loss of Ab binding without KO, preserving physiologic protein expression, regulation, and intracellular signaling. Critically, this strategy enables targeting one or more genes fundamental for leukemia survival, resulting in potent anti-leukemia efficacy with minimal on-target/off-tumor toxicity. Methods. Through epitope-mapping, we identified substitutions in the FLT3, KIT and CD123 extracellular-domains that avoid detection by therapeutic Abs. We validated the functionality of mutated receptors (ligand affinity, western-blot, proliferation, RNAseq, phospho-proteomic MS) and their resistance to on-target killing (mAb-affinity, CAR-T co-culture). We optimized a base-editing protocol to introduce these mutations in CD34+HSPCs and developed advanced in vivo models with co-engraftment of healthy HSPCs, patient-derived AML xenografts (PDX) and CAR-T to assess selective elimination of leukemia and protection of healthy hematopoiesis. Results. To develop our approach, we selected mAbs under development for AML therapy: clone 4G8 (FLT3), Fab-79D (KIT) and 7G3 (CD123). To identify residues involved in mAb binding, we designed Sleeping Beauty epitope mapping libraries. We found that single amino-acid substitutions can disrupt therapeutic mAb binding despite preserved surface receptor. Since epitope engineering can be achieved by point mutations, we reasoned that base editing (BE) could be a suitable and safer option compared to homology directed repair (HDR). By electroporating sgRNA+ABE variants, we achieved successful epitope editing with minimal toxicity. By using fluorescent FLT3L, SCF and IL-3, we confirmed preserved ligand binding to edited receptors. Activation of downstream signaling was confirmed by western blot. By performing in vitro killing assays, we found that, while cells expressing WT FLT3, KIT or CD123 were eliminated, those expressing epitope-edited variants were resistant to CAR-mediated killing and survived up to experiment termination without eliciting T cell activation and degranulation. To introduce our variants into human HSPCs, we optimized a BE protocol on mobilized peripheral blood-derived CD34+ cells, achieving up to 86%, 78% and 78% efficiency for FLT3, KIT and CD123, respectively. Contrary to previous observations with HDR editing, BE efficiencies were similar in bulk and primitive, HSC-enriched subsets (CD90+45RA-) with no skewing of stem phenotype. BE HSPCs were resistant to CAR or mAb-mediated killing in vitro. To confirm the safety of our approach, we compared FLT3, CD123, KIT edited to AAVS1 control HSPCs and found no differences in proliferative response, transcriptional changes (RNAseq), phospho-proteomic profile and colony-forming capacity. Xenotransplantation of BE HSPCs in NBSGW mice showed preserved repopulation and multilineage differentiation capacity, both in primary and secondary recipients. To assess if FLT3 CAR can eliminate AML while sparing FLT3-edited hematopoiesis, we sequentially engrafted NBSGW mice with HSPCs and human PDX cells. We observed a significant increase in the percentage of FLT3 edited cells in CAR-treated mice and relative depletion of CD19+ subsets (pre-B, pro-B), granulocytes, granulo-mono progenitors (GMP) and lymphoid-primed multipotent progenitors (LMPP) only in the AAVS1-BE group, while mice engrafted with FLT3-BE HPSC were protected. Concomitantly, mice treated with 4G8-CAR achieved complete AML eradication. CAR-T exposed to FLT3-BE hematopoiesis displayed lower PD-1 expression compared to AAVS1-BE. As done for FLT3, we transplanted CD123-BE HSPCs and confirmed multilineage repopulation comparable to controls. Mice treated with CD123 CAR-T showed eradication of AML cells and concomitant protection of epitope-edited myeloid lineages, including granulocytes, DCs and HSPCs. To test whether our approach allows multiplexing, we tested combinations of FLT3, KIT and CD123 editing, which provided additive protection from triple-specific CAR-T cells in vitro. Furthermore, combined dual FLT3+CD123 BE could protect hematopoietic lineages in vivo when mice were treated with FLT3+CD123 CAR-T, which in turn were able to eradicate PDXs resistant to FLT3-targeting alone. Discussion. Our studies provide proof of concept that tumor-associated Ags shared by normal tissue can be safely targeted by precisely modifying the epitope recognized by adoptive immunotherapies in healthy cells, endowing them with selective resistance and generating an artificial leukemia-restricted Ag. The innovative tools developed in this work can increase the therapeutic index of AML immunotherapies and enable long-term anti-leukemia maintenance. By restricting on-target activity to leukemia cells, epitope-editing can reduce the Ag burden to which CAR-T are exposed, decreasing undesired CAR-T stimulation, cytokine secretion and exhaustion. Epitope editing can easily be multiplexed to enable combination therapies while avoiding overlapping toxicities, further enhancing the chances for tumor eradication. Finally, epitope editing may be exploited to improve non-genotoxic conditioning for autologous gene therapy or HSCT, either alone or in combination with other therapeutic targets, to avoid depletion of transplanted cells and achieve in vivo selection of genome-engineered cells. Conclusion. We believe that epitope-engineering of HSPCs is a novel and highly promising technology that can enable safer and more effective immunotherapies when on-target/off-tumor toxicities are the key limiting factor to successful clinical translation. Citation Format: Gabriele Casirati, Andrea Cosentino, Adele Mucci, Mohammed S. Mahmoud, Iratxe Ugarte Zabala, Jing Zeng, Scott B. Ficarro, Denise Klatt, Christian Brendel, Alessandro Rambaldi, Jerome Ritz, Jarrod A. Marto, Danilo Pellin, Daniel E. Bauer, Scott A. Armstrong, Pietro Genovese. Epitope editing enables targeted immunotherapy of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr NG05.

A bupropion modulatory site in Gloeobacter violaceus ligand-gated ion channel

Bupropion is an atypical antidepressant and smoking cessation drug that causes adverse effects such as insomnia, irritability, and anxiety. Bupropion inhibits dopamine and norepinephrine reuptake transporters and eukaryotic cation-conducting pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine (nACh) and serotonin type 3A (5-HT3A) receptors, at clinically relevant concentrations. Here, we demonstrate that bupropion also inhibits a prokaryotic homolog of pLGICs, the Gloeobacter violaceus ligand-gated ion channel (GLIC). Using GLIC as a model, we used molecular docking to predict binding sites for bupropion. Several clusters of bupropion binding pose within the transmembrane domain were identified, with the predominant cluster being localized to the interface between transmembrane segments M1 and M3 of adjacent subunits. Residues W213, T214 and W217 in the first transmembrane segment, M1, and F267 and I271 in the third transmembrane segment, M3, most frequently reside within 4 Å distance from bupropion. We then used single amino acid substitutions at these positions and two-electrode voltage-clamp recordings to determine their impact on bupropion inhibitory effects. The substitution T214F alters bupropion potency by shifting the IC50 to a 13-fold higher value compared to wild-type GLIC. Residue T214 is found within a previously identified binding pocket for neurosteroids and lipids in GLIC. This intersubunit binding pocket is structurally conserved and almost identical to a binding pocket described for neurosteroids in GABAA receptors. Our data thus suggest that T214 that lines a previously identified lipophilic binding pocket in GLIC and GABAA receptors is also a modulatory site for bupropion interaction with GLIC.

Abstract 1838: Advancing cancer research with Next-Generation Protein Sequencing™ on Platinum™ by Quantum-Si   

Abstract Protein sequencing has the potential of playing a crucial role in advancing cancer research by providing insights into the specific proteins involved in cancer pathways. New discoveries from protein sequencing advance our understanding of cancer and potential targeted therapies. Here, we demonstrate the power of next-generation protein sequencing™ using the insight-generating Platinum™ platform. Individual peptides are digested from proteins and immobilized on a semiconductor chip and probed by dye-labeled N-terminal amino acid (NAA) recognizers, followed by subsequent aminopeptidase cleavage to expose each NAA in the peptide for recognition. By recording and analyzing fluorescent intensity, lifetime, and binding kinetics of each NAA binding event, we can decipher single amino acid substitutions and modifications. To demonstrate the versatility of this methodology across proteins associated with cellular biology, we sequenced a series of interleukins: IL4, IL6, IL7, IL13, IL20, and IL34. Interleukins, a group of cytokines that play a vital role in cancer by modulating immune responses, promoting inflammation, and influencing tumor microenvironment, impacting tumor growth and the efficacy of treatments. We have successfully sequenced all the mentioned interleukins individually or within protein mixtures. Furthermore, we were able to sequence IL6 that was enriched through immunoprecipitation from human serum. Finally, we demonstrated the unique potential of Platinum in detecting post-translational modifications (PTMs) relevant to cancer, as exemplified by the successful identification of arginine citrullination in vimentin, an onco-protein that promotes cancer cell mobility and invasion into surround tissues. These results demonstrate the transformative potential of next-generation protein sequencing using Platinum™ in enabling the identification of proteins in complex mixtures and biofluids, as well as the detection of critical PTMs implicated in human pathophysiology, offering valuable insights into cancer research. Citation Format: Khanh Dinh Nguyen. Advancing cancer research with Next-Generation Protein Sequencing™ on Platinum™ by Quantum-Si    [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1838.

Abstract 377: Saturation mutagenesis of KEAP1 to identify pathogenic mutations in lung cancer patients and gain insight into KEAP1 molecular function

Abstract Introduction: KEAP1 and NFE2L2 (NFR2) mutations occur in roughly 20% of non-small cell lung cancer (NSCLC) patients and are associated with resistance to a broad range of therapies including radiotherapy, chemotherapy, targeted therapies, and immunotherapy. In prior work, we demonstrated that not all KEAP1 mutations observed in NSCLC patients are pathogenic. These benign mutations have a wild-type phenotype that does not impart therapy resistance. However, only roughly 100 of ~13,000 (&amp;lt;1%) possible KEAP1 single amino acid substitution mutations have been characterized. Here, we performed saturation mutagenesis of KEAP1 to comprehensively evaluate all possible point mutation phenotypes in vitro. Methods: Saturation mutagenesis was accomplished using the High-throughput Mutagenesis by Integrated TilEs (MITE) method. In brief, the KEAP1 sequence was divided into 21 tiles each containing a 90 bp variable region flanked by a 30 bp constant region. At each codon position, mutations representing the 20 possible natural amino acids and a stop codon were included for a total of 13,076 KEAP1 variants. The tiles were assembled such that each full length KEAP1 construct contained a single desired mutation. The KEAP1 MITE lentivirus library was transduced into H1299-KEAP1NULL cells that were then treated with vehicle or H2O2 for 24 days. The KEAP1 ORF was amplified from gDNA and mutations were quantified by next generation sequencing. Mutation enrichment analysis was used to derive functional annotations. Results: Oxidative stress resulted in a bimodal distribution of KEAP1 mutations. Cells with KEAP1 nonsense mutations were enriched while cells with silent mutations were depleted. Using a threshold of 95% specificity for silent mutations to distinguish benign from pathogenic mutations, 94% of nonsense mutations and 88% of previously characterized pathogenic mutations were identified as pathogenic. While the majority of KEAP1 mutations were benign (64%), analysis of mutations found in TCGA NSCLC patients showed an enrichment of pathogenic mutations (72%). Overlaying mutation calls onto the KEAP1 structure showed enrichment of pathogenic mutations in the BTB homodimerization interface, hydrophobic core of the oxidative stress sensing IVR domain, and NRF2 binding pocket of the Kelch domain. Conclusions: Saturation mutagenesis of KEAP1 generated a comprehensive list of pathogenic KEAP1 single amino acid substitutions. Assignments strongly agreed with biologic function and prior literature. The assessment of KEAP1 mutation phenotypes is important as benign mutations occur in a significant subset of NSCLC patients and are not expected to impart therapy resistance. Our results will allow identification of benign and pathogenic KEAP1 mutations in clinical studies, which will enable testing of personalized therapeutic strategies for patients with KEAP1 mutations. Citation Format: Noah Kastelowitz, Shashank Shrishrimal, Soyeong Jun, Anni M. Zhang, Rui Wang, Ilayda Ilerten, Maximilian Diehn. Saturation mutagenesis of KEAP1 to identify pathogenic mutations in lung cancer patients and gain insight into KEAP1 molecular function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 377.

Abstract 4899: Interface-guided phenotyping of coding variants in the transcription factor RUNX1

Abstract Understanding the consequences of single amino acid substitutions in cancer driver genes remains an unmet need. Perturb-seq provides a tool to investigate the effects of individual mutations on cellular programs. Here we deploy SEUSS, a Perturb-seq like approach, to generate and assay mutations at physical interfaces of the RUNX1 Runt domain. We measured the impact of 115 mutations on RNA profiles in single myelogenous leukemia cells and used the profiles to categorize mutations into three functionally distinct groups: wild-type (WT)-like, loss-of-function (LOF)-like and hypomorphic. Notably, the largest concentration of functional mutations (non-WT-like) clustered at the DNA binding site and contained many of the more frequently observed mutations in human cancers. Hypomorphic variants shared characteristics with loss of function variants but had gene expression profiles indicative of response to neural growth factor and cytokine recruitment of neutrophils. Additionally, DNA accessibility changes upon perturbations were enriched for RUNX1 binding motifs, particularly near differentially expressed genes. Overall, our work demonstrates the potential of targeting protein interaction interfaces to better define the landscape of prospective phenotypes reachable by amino acid substitutions. Citation Format: Kivilcim Ozturk, Rebecca Panwala, Jeanna Sheen, Kyle Ford, Nathan Jayne, Dong-Er Zhang, Stephan Hutter, Torsten Haferlach, Trey Ideker, Prashant Mali, Hannah Carter. Interface-guided phenotyping of coding variants in the transcription factor RUNX1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4899.

Abstract 556: The Idylla™ IDH1-2 Mutation Assay Kit: A tool for mutation detection in IDH1 and IDH2 genes

Abstract Background: Mutations in the genes encoding isocitrate dehydrogenase (IDH1 and IDH2) are frequently detected in various cancers, including acute myeloid leukemia (~20%) and glioma (up to ~80%). They commonly involve a single amino acid substitution at codon R132 in IDH1, and R140 or R172 in IDH2 and are important for diagnosis and prognosis as well as therapeutic decisions. With advancements in molecular diagnostics and precision therapy, IDH inhibitors such as ivosidenib, vorasidenib and enasidenib are now mainstay in management of patients with a susceptible mutation. Methods: The Idylla™ IDH1-2 Mutation Assay Kit (RUO) is a fully automated qPCR assay for the qualitative detection of 15 common mutations in IDH1 (R132C/H/G/S/L) and IDH2 (R140Q/L/G/W, R172K/M/G/W/S) at codon level. The assay consists of a ready-for-use Idylla™ DNA Cartridge and an assay specific Idylla™ Vial. It runs on the Idylla™ Platform and covers the entire process from sample to result including specimen processing, real-time PCR amplification, detection, data analysis, and result reporting, within 100 minutes.Five clinical sites in US and Europe participated in an early access program to evaluate performance of the assay and establish a final classification algorithm by testing a total of 241 specimens, including FFPE, human whole blood and bone marrow samples, and extracted DNA from these sample types. Reference results were obtained using a range of established methods with different mutations in scope, such as Sanger sequencing, immunohistochemistry, NGS and qPCR. Results: Of 241 Cartridges, one invalid result was obtained, while 13 results were flagged as having low DNA input. Compared to available reference methods, overall concordance was 98.3% (235/239), 100% (140/140) and 99.6% (230/231) for IDH1 R132, IDH2 R140 and R172, respectively (see table). Conclusion: The Idylla™ IDH1-2 Mutation Assay Kit demonstrates a high concordance with established reference methods across a range of different specimen types. Reference method(s) PPA (%) NPA (%) OPA (%) MUT WT Total Idylla™ IDH1-2 Mutation Assay Kit IDH1_R132x 111 3 114 99.1 97.6 98.3 WT 1 124 125 Total 112 127 239 IDH2_R140x 10 0 10 100.0 100.0 100.0 WT 0 130 130 Total 10 130 140 IDH2_R172x 25 0 25 96.2 100.0 99.6 WT 1 205 206 Total 26 205 231 Total 148 462 610 Citation Format: Bram De Craene, Katrien Trappeniers, Ana Marcelino, Lee Robertson, Phillippe Taniere, Jo Van Dorpe, Malaïka Van der Linden, Maria Arcila, Khedoudja Nafa, Jochen K. Lennerz, Richard Craig Carson, Richard D. Ledding, Sofie Metsu. The Idylla™ IDH1-2 Mutation Assay Kit: A tool for mutation detection in IDH1 and IDH2 genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 556.

Cross-neutralizing antibody against emerging Omicron subvariants of SARS-CoV-2 in infection-naïve individuals with homologous BNT162b2 or BNT162b2(WT + BA.4/5) bivalent booster vaccination

Since the emergence of SARS-CoV-2, different variants and subvariants successively emerged to dominate global virus circulation as a result of immune evasion, replication fitness or both. COVID-19 vaccines continue to be updated in response to the emergence of antigenically divergent viruses, the first being the bivalent RNA vaccines that encodes for both the Wuhan-like and Omicron BA.5 subvariant spike proteins. Repeated infections and vaccine breakthrough infections have led to complex immune landscapes in populations making it increasingly difficult to assess the intrinsic neutralizing antibody responses elicited by the vaccines. Hong Kong’s intensive COVID-19 containment policy through 2020–2021 permitted us to identify sera from a small number of infection-naïve individuals who received 3 doses of the RNA BNT162b2 vaccine encoding the Wuhan-like spike (WT) and were boosted with a fourth dose of the WT vaccine or the bivalent WT and BA.4/5 spike (WT + BA.4/5). While neutralizing antibody to wild-type virus was comparable in both vaccine groups, BNT162b2 (WT + BA.4/BA.5) bivalent vaccine elicited significantly higher plaque neutralizing antibodies to Omicron subvariants BA.5, XBB.1.5, XBB.1.16, XBB.1.9.1, XBB.2.3.2, EG.5.1, HK.3, BA.2.86 and JN.1, compared to BNT162b2 monovalent vaccine. The single amino acid substitution that differentiates the spike of JN.1 from BA.2.86 resulted in a profound antigenic change.

Fit for Purpose Approach To Evaluate Detection of Amino Acid Substitutions in Shotgun Proteomics.

Amino acid substitutions (AASs) alter proteins from their genome-expected sequences. Accumulation of substitutions in proteins underlies numerous diseases and antibiotic mechanisms. Accurate global detection of AASs and their frequencies is crucial for understanding these mechanisms. Shotgun proteomics provides an untargeted method for measuring AASs but introduces biases when extrapolating from the genome to identify AASs. To characterize these biases, we created a "ground-truth" approach using the similarities betweenEscherichia coli and Salmonella typhimurium to model the complexity of AAS detection. Shotgun proteomics on mixed lysates generated libraries representing ∼100,000 peptide-spectra and 4161 peptide sequences with a single AAS and defined stoichiometry. Identifying S. typhimurium peptide-spectra with only the E. coli genome resulted in 64.1% correctly identified library peptides. Specific AASs exhibit variable identification efficiencies. There was no inherent bias from the stoichiometry of the substitutions. Short peptides and AASs localized near peptide termini had poor identification efficiency. We identify a new class of "scissor substitutions" that gain or lose protease cleavage sites. Scissor substitutions also had poor identification efficiency. This ground-truth AAS library reveals various sources of bias, which will guide the application of shotgun proteomics to validate AAS hypotheses.

Novel electroporation-based genome editing of carnation plant tissues using RNPs targeting the anthocyanidin synthase gene.

A novel electroporation method for genome editing was performed using plant tissue samples by direct RNPs-introduction in carnation. Genome editing is becoming a very useful tool in plant breeding. In this study, a novel electroporation method was performed for genome editing using plant tissue samples. The objective was to create a flower color mutant using the pink-flowered carnation 'Kane Ainou 1-go'. For this purpose, a ribonucleoprotein consisting of guide RNA and clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) was introduced into the stem tissue to induce mutations in the anthocyanidin synthase (ANS) gene, which is involved in anthocyanin biosynthesis. As the ANS of 'Kane Ainou 1-go' has not been previously isolated, we initially isolated the ANS gene from 'Kane Ainou 1-go' for characterization. Southern hybridization analysis confirmed that the ANS gene was present in the genome as a two-allele gene with a pair of homologous sequences (ANS-1 and 2); these sequences were used as the target for genome editing. Genome editing was performed by introducing #2_single-guide RNA into the stem tissue using the ribonucleoprotein. This molecule was used because it exhibited the highest efficiency in an analysis of cleavage activity against the target sequence in vitro. Cleaved amplified polymorphic sequence analysis of genomic DNA extracted from 85 regenerated individuals after genome editing was performed. The results indicated that mutations in the ANS gene may have been introduced into two lines. Cloning of the ANS gene in these two lines confirmed the introduction of a single nucleotide substitution mutation for ANS-1 in both lines, and a single amino acid substitution in one line. We discussed the possibility of color change by the amino acid substitution, and also the future applications of this technology.

Tuning Supramolecular Chirality in Iodinated Amphiphilic Peptides Through Tripeptide Linker Editing.

Protease-cleavable supramolecular oligopeptide nanofilaments are promising materials for targeted therapeutics and diagnostics. In these systems, single amino acid substitutions can have profound effects on the supramolecular structure and consequent proteolytic degradation, which are critical parameters for their intended applications. Herein, we describe changes to the self-assembly and proteolytic cleavage of iodine containing sequences for future translation into matrix metalloprotease (MMP-9)-activated supramolecular radio-imaging probes. We use a systematic single amino acid exchange in the tripeptide linker region of these peptide amphiphiles to provide insights into the role of each residue in the supramolecular assemblies. These modifications resulted in dramatic changes in the nature of the assembled structures formed, including an unexpected chiral inversion. By using circular dichroism, atomic force microscopy, Fourier transform infrared spectroscopy, and molecular dynamics simulations, we found that the GD loop, a common motif in β-turn elements, induced a reversal of the chiral orientation of the assembled nanofibers. In addition to the impact on peptide packing and chirality, MMP-9-catalyzed hydrolysis was evaluated for the four peptides, with the β-sheet content found to be a stronger determinant of enzymatic hydrolysis than supramolecular chirality. These observations provide fundamental insights into the sequence design in protease cleavable amphiphilic peptides with the potential for radio-labeling and selective biomedical applications.