Increasing use of covalent and noncovalent inhibitors of Bruton’s tyrosine kinase (BTK) has elucidated a series of acquired drug-resistant BTK mutations in patients with B cell malignancies. Here we identify inhibitor resistance mutations in BTK with distinct enzymatic activities, including some that impair BTK enzymatic activity while imparting novel protein-protein interactions that sustain B cell receptor (BCR) signaling. Furthermore, we describe a clinical-stage BTK and IKZF1/3 degrader, NX-2127, that can bind and proteasomally degrade each mutant BTK proteoform, resulting in potent blockade of BCR signaling. Treatment of chronic lymphocytic leukemia with NX-2127 achieves >80% degradation of BTK in patients and demonstrates proof-of-concept therapeutic benefit. These data reveal an oncogenic scaffold function of mutant BTK that confers resistance across clinically approved BTK inhibitors but is overcome by BTK degradation in patients.

Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies remains challenging in part due to the lack of faithful preclinical in vitro models. Here we establish twelve patient-derived WT cell lines and demonstrate that these models faithfully recapitulate WT biology using genomic and transcriptomic techniques. We then perform loss-of-function screens to identify the nuclear export gene, XPO1, as a vulnerability. We find that the FDA approved XPO1 inhibitor, KPT-330, suppresses TRIP13 expression, which is required for survival. We further identify synergy between KPT-330 and doxorubicin, a chemotherapy used in high-risk FHWT. Taken together, we identify XPO1 inhibition with KPT-330 as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo.

Immunocompromised patients with coronavirus disease 2019 were prospectively enrolled from March to November 2022 to understand the association between antibody responses and severe acute respiratory syndrome coronavirus 2 shedding. A total of 62 patients were analyzed, and the results indicated a faster decline in genomic and subgenomic viral RNA in patients with higher neutralizing and S1-specific immunoglobulin G (IgG) antibodies (both P < .001). Notably, high neutralizing antibody levels were associated with a significantly faster decrease in viable virus cultures (P = .04). Our observations suggest the role of neutralizing antibodies in prolonged virus shedding in immunocompromised patients, highlighting the potential benefits of enhancing their humoral immune response through vaccination or monoclonal antibody treatments.

Copy number variation (CNV) of samples in TCGA

DEDD overexpression confers resistance to TNFα mediated apoptosis.

Therapy resistance is a major challenge in the treatment of cancer. Here, we performed CRISPR/Cas9 screens across a broad range of therapies used in acute myeloid leukemia to identify genomic determinants of drug response. Our screens uncovered a selective dependency on RNA splicing factors whose loss preferentially enhanced response to the BCL2 inhibitor venetoclax. Loss of the splicing factor RBM10 augmented response to venetoclax in leukemia yet was completely dispensable for normal hematopoiesis. Combined RBM10 and BCL2 inhibition led to mis-splicing and inactivation of the inhibitor of apoptosis XIAP and downregulation of BCL2A1, an anti-apoptotic protein implicated in venetoclax resistance. A novel inhibitor of splicing kinase families CLKs and DYRKs led to aberrant splicing of key splicing and apoptotic factors that synergized with venetoclax and overcame resistance to BCL2 inhibition. Our findings underscore the importance of splicing in modulating response to therapies and provide a strategy to improve venetoclax-based treatments. Citation Format: Eric Wang, Omar Abdel-Wahab, Robert K Bradley, Jose Mario Bello, Won Jun Kim, Carine Bossard. Modulation of RNA splicing enhances response to BCL2 inhibition in leukemia [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A04.

•Immunocompromised patients with COVID-19 shed viable virus for median 4 weeks.•B-cell depleting therapy increases the risk of prolonged viable viral shedding.•Vaccination shortens the viable virus shedding in immunocompromised patients.

Dear Editor, During the coronavirus disease 2019 (COVID-19) pandemic of more than three years, several variants have evolved from the previously prevalent strains, being categorized as variants of concern (VOCs), variants of interest (VOIs), variants of high consequence and variants being monitored.1 The origin of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants is unclear, but one possible explanation is that they stem from immunocompromised patients.2 Whole-genome sequencing (WGS) is a useful tool for detecting new mutations and emerging SARS-CoV-2 variants.3 Here, we used WGS to investigate the features of nonsynonymous SARS-CoV-2 mutations that appeared in immunocompromised patients with persistent viral detection during the Omicron-prevalent era. This prospective study was conducted at a 2732-bed tertiary teaching hospital from February to November 2022. We enrolled immunocompromised adults within 12 weeks of initial SARS-CoV-2 diagnosis and gathered nasopharyngeal swabs, saliva and blood samples on a weekly basis. We also performed real-time reverse transcription-polymerase chain reaction tests for SARS-CoV-2, viral cultures, plaque reduction neutralization tests, and WGS on at least two serial samples from each patient. The details of patient enrollment, sample collection, and laboratory procedures are explained in the Supporting Information. A total of 37 WGS results from 13 SARS-CoV-2 patients were included in the final analysis (Figure S1). The baseline features of the study subjects are described in Table S1. The WGS analysis results were obtained from each immunocompromised patient with a median frequency of three times (interquartile range [IQR] 2–3). The median interval between consecutive WGS analyses was 20 days (IQR 15–46 days). The patients acquired a median of two nonsynonymous mutations (IQR 1–7), excluding temporary mutations. The specific mutations compared with the Wuhan Hu-1 reference genome and the acquired mutations in subsequent WGS are presented for each patient in Figure 1 and Table 1. Among the total 87 nonsynonymous mutations, 16 (18.4%) and 13 (14.9%) mutations were classified as persistent and temporary mutations, respectively. More than half of the mutations were detected in the ORF1ab region (Figure S2). There were 29 mutations in the S region, 12 of which were associated with immune evasion (see Supporting Information). Also, 13 mutations in the ORF1ab, S, and M regions were the defining mutations of the major variants, including Omicron BA.1, BA.2.75, BA.4/5, and several XBB subvariants,4 and eleven of these mutations occurred in the S region (Table 2). The proportion of acquired mutations that were defining mutations of other variants was higher in the S region (11/29, 37.9%) than in the whole genomic region (13/87, 14.9%). Table S2 outlines the number of nonsynonymous mutations associated with immune evasion and the defining mutations of the major variants for each patient. The V792I mutation in the nsp12, also known as V5184I in the ORF1ab region, is reported to be associated with viral resistance to remdesivir.5 Patient H acquired this mutation 142 days after SARS-CoV-2 diagnosis. Before the acquisition of this mutation, the patient had prolonged exposures to remdesivir, dexamethasone, and baricitinib for 28, 17 and 15 days, respectively (Figure 2). This patient also received high-dose steroids (≥ equivalent doses of prednisolone 0.3 mg/kg daily) for more than two months. While B-cell depletion is considered the main factor affecting the period of SARS-CoV-2 shedding,6 the presence of high neutralizing antibody titers does not always ensure eradication of SARS-CoV-2 infection.7 Patient J shed the virus persistently from days 72–79 despite maintaining a high titer of neutralizing antibodies since the initial COVID-19 diagnosis (Figure 2). WGS analyses were conducted on days 79 and 98, and the missense mutation S:L452Q, detected on day 79, seemed to have ‘reverted’ by day 98. This mutation has been reported to be associated with immune evasion and a decreased sensitivity to neutralizing antibodies.8 While we could not determine the exact duration of the presence of the S:L452Q mutation or assess the status of T-cell immunity for this patient, persistent viral shedding might be attributed to this mutation and its diminished sensitivity to neutralizing antibodies. This study investigated the dynamics and characteristics of SARS-CoV-2 mutations in immunocompromised patients with persistent viral detection during the Omicron era. Each patient acquired a median of two amino acid substitutions over a median of 51 days, which equals 14.2 substitutions per year. In comparison, other studies from the pre-Omicron era reported nonsynonymous mutation rates of 24.4–52.4 substitutions per year for immunocompromised patients.7, 9, 10 While our study involved a larger cohort of such patients, some exhibited a milder immunocompromised status than those in previous studies, potentially leading to variations in the mutation rates. Several mutations seem to have emerged sporadically, distributed throughout the whole SARS-CoV-2 genome. This distribution pattern mirrors findings from an earlier study that also reported a sporadic distribution of various mutations across the SARS-CoV-2 genome in immunocompromised individuals.2, 7, 9, 10 The ORF1ab region, accounting for up to 21,290 nucleotides (71.2%) of the 29,900 total, housed more than half of the nonsynonymous mutations identified in this study. The S region, consisting of 3,822 nucleotides (12.8%), harboured about one-third of the mutations. The adjusted mutation numbers per kilobase were 2.1 for the ORF1ab region and 7.6 for the S region. Additionally, mutations known to contribute to immune escape, or those defining other variants designated as VOIs or VOCs, primarily arose in the S region. This observation aligns with findings from other studies.7, 9 The immunocompromised patients in this study were predominantly infected with BA.2 or BA.2.3 sub-lineages. We identified mutations typical of BA.4/5, BA.2.75, BQ.1 and various XBB subvariants in the SARS-CoV-2 genomes from these patients. Notably, during the pre-Omicron era, immunosuppressed patients were found to acquire nonsynonymous mutations linked to subsequent SARS-CoV-2 lineages.2, 7, 9 These observations suggest that persistent viral infections in immunocompromised patients could drive the acquisition of new mutations, leading to the adaptive evolution of SARS-CoV-2. Therefore, tracking these mutations might provide insights into viral adaptation and the advent of new SARS-CoV-2 variants. This study has several limitations. Viral evolution within populations can be influenced by infection prevalence and immune landscapes, as well as ethnic genetic predispositions.11 Our study was conducted during the Omicron-prevalent era, and the lack of data from the pre-Omicron period limits the generalization of our findings. Also, the patients in our study might not fully represent the spectrum of immunity statuses in immunocompromised patients. Specifically, eleven out of the thirteen patients in our study had hematologic malignancies, and eight had not received SARS-CoV-2 vaccines. This particular immunity profile could give rise to mutations distinct from those observed in other immunocompromised populations.11 Consequently, there might be some potential for regional or immunological biases in our findings. In conclusion, during the Omicron-prevalent era, SARS-CoV-2 genomes of immunocompromised individuals with persistent viral detection exhibited several mutations. These mutations have been reported to be associated with immune evasion, remdesivir resistance and new variant emergence. Given the rise of new subvariants with mutations associated with immune evasion or remdesivir resistance and the potential for immunocompromised individuals to shed viable viruses, decisions regarding the termination of isolation for immunocompromised patients with SARS-CoV-2 infection should be approached with caution. The authors declare no conflict of interest. Korea National Institute of Health, Grant/Award Numbers: 2022-ER1609-00, 2022-NI-043-00 and 6634-325-210; Ministry of Science and Information & Communications Technology, Republic of Korea, Grant/Award Number: NRF-2022M3A9I2017241; Ministry of Education, Republic of Korea, Grant/Award Number: 2021R1A6C101C570 All data supporting the findings of this study are available within the paper and its supplementary material and from the corresponding authors upon reasonable request. 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.

Abstract Background Immunocompromised patients have been shown to have prolonged SARS-CoV-2 viral shedding. However, there are limited data on the longitudinal association between immune response and viable virus shedding in immunocompromised patients. Thus, we aimed to investigate the relationship between the kinetics of the immune responses and the duration of viable virus shedding in immunocompromised patients. Methods We prospectively enrolled immunocompromised patients with COVID-19 who were admitted to a tertiary center in Seoul, South Korea, from March 2022 to August 2022. SARS-CoV-2 S1-specific IgG antibody and neutralizing antibody were measured by ELISA and plaque reduction neutralizing assay, respectively. Genomic RNA, subgenomic RNA, and culture-based virus isolation were performed on respiratory samples to identify viral shedding. Results A total of 62 patients whose serial blood and respiratory samples were obtained were analyzed. Significant group (degree of antibody response)-by-time (interval from the infection) interaction was observed in terms of both S1-IgG antibody (P < 0.001) and neutralizing antibody (P < 0.001), that is, the genomic RNA declined significantly more rapidly in patients with higher antibody response compared with lower antibody response. There was a significant difference in the proportion of culturable virus according to the time from SARS-CoV-2 infection depending on neutralizing antibody level (P=0.04), while there was no difference depending on the S1-IgG antibody level (P=0.06).Figure 1.SARS-CoV-2 viral shedding and the degree of S1-specific IgG antibody according to the time from the diagnosisFigure 2.Genomic viral RNA shedding by time from the SARS-CoV-2 infection depending on the degree of antibody responses. Left panel. S1-IgG antibody Right panel. Neutralizing antibodyFigure 3.Proportion of culturable virus shedding by time from the SARS-CoV-2 infection depending on the degree of antibody responses. Conclusion Our findings suggest that neutralizing antibody response is an important factor associated with prolonged viable SARS-CoV-2 shedding in immunocompromised patients with COVID-19. These findings provide us important insight into the pathophysiology of viral clearance and the potential role of boosting humoral immune response against COVID-19 in immunocompromised patients including vaccination and monoclonal antibody prophylaxis or therapy.Table 1.Baseline characteristics of the study participants. Data represents n(%) unless otherwise indicated aRituximab or bispecific T-cell engager b≥0.3 mg/kg corticosteroids for ≥3 weeks in the past 60 days cNational Institue of Health (NIH) COVID-19 severity classification dRemdesivir or nirmatrelvir/ritonavir within 5 days of diagnosis Disclosures All Authors: No reported disclosures

Supplementary Figure from Impaired Proteolysis of Noncanonical RAS Proteins Drives Clonal Hematopoietic Transformation

DEDD and MCL1 overexpression are not necessary for initiation of tumorigenesis.

Sequences for arrayed RNAi vectors used

DEDD overexpression does not lead to resistance of the intrinsic pathway of apoptosis.

There are limited data supporting current Centers for Disease Control and Prevention guidelines for the isolation period in moderate to severely immunocompromised patients with coronavirus disease 2019 (COVID‐19). Adult COVID‐19 patients who underwent solid organ transplantation (SOT) or received active chemotherapy against hematologic malignancy were enrolled and weekly respiratory samples were collected. Samples with positive genomic real‐time polymerase chain reaction results underwent virus culture and rapid antigen testing (RAT). A total of 65 patients (40 with hematologic malignancy and 25 SOT) were enrolled. The median duration of viable virus shedding was 4 weeks (interquartile range: 3–7). Multivariable analysis revealed that B‐cell depletion (hazard ratio [HR]: 4.76) was associated with prolonged viral shedding, and COVID‐19 vaccination (≥3 doses) was negatively associated with prolonged viral shedding (HR: 0.22). The sensitivity, specificity, positive predictive value, and negative predictive value of RAT for viable virus shedding were 79%, 76%, 74%, and 81%, respectively. The negative predictive value of RAT was only 48% (95% confidence interval [CI]: 33–65) in the samples from those with symptom onset ≤20 days, but it was as high as 92% (95% CI: 85–96) in the samples from those with symptom onset >20 days. About half of immunocompromised COVID‐19 patients shed viable virus for ≥4 weeks from the diagnosis, and virus shedding was prolonged especially in unvaccinated patients with B‐cell‐depleting therapy treatment. RAT beyond 20 days in immunocompromised patients had a relatively high negative predictive value for viable virus shedding.

Ongoing emergence of SARS-CoV-2 Omicron subvariants and their rapid worldwide spread pose a threat to public health. From November 2022 to February 2023, newly emerged Omicron subvariants, including BQ.1.1, BF.7, BA.5.2, XBB.1, XBB.1.5, and BN.1.9, became prevalent global strains (>5% global prevalence). These Omicron subvariants are resistant to several therapeutic antibodies. Thus, the antiviral activity of current drugs such as remdesivir, molnupiravir, and nirmatrelvir, which target highly conserved regions of SARS-CoV-2, against newly emerged Omicron subvariants need to be evaluated. We assessed the antiviral efficacy of the drugs using the half-maximal inhibitory concentration (IC50) against human isolates of 23 Omicron subvariants and four former SARS-CoV-2 variants of concern (VOCs) and compared it with the antiviral efficacy of these drugs against the SARS-CoV-2 reference strain (hCoV/Korea/KCDC03/2020). Maximal IC50-fold changes of remdesivir, molnupiravir, and nirmatrelvir were 1.9 (BA.2.75.2), 1.2 (B.1.627.2), and 1.4 (BA.2.3), respectively, compared to median IC50 values of the reference strain. Moreover, median IC50-fold changes of remdesivir, molnupiravir, and nirmatrelvir against the Omicron variants were 0.96, 0.4, and 0.62, respectively, similar to the 1.02, 0.88, and 0.67, respectively, median IC50-fold changes for previous VOCs. Although K90R and P132H in Nsp 5, and P323L, A529V, G671S, V405F, and ins823D in Nsp 12 mutations were identified, these amino acid substitutions did not affect drug antiviral activity. These results indicate that current antivirals retain antiviral efficacy against newly emerged Omicron subvariants. It is important to continue active surveillance and testing of new variants for drug resistance to enable early identification of drug-resistant strains.

Despite a high vaccination rate, the COVID-19 pandemic continues with immune-evading Omicron variants. The success of additional antigenic stimulation through breakthrough infection (BI) and updated vaccination in overcoming antigenic imprinting needs to be determined. Participants in a long-term follow-up cohort of healthcare worker (HCW) vaccinee were categorized according to their infection/vaccination status. Anti-SARS-CoV-2 spike/nucleocapsid protein antibodies were measured, and plaque reduction neutralization tests (PRNTs) against wild-type (WT), BA.5, BN.1, and XBB.1.5 were conducted. The neutralization activity of intravenous immunoglobulin (IVIG) products was evaluated to assess the immune status of the general population. Ninety-five HCWs were evaluated and categorized into seven groups. The WT PRNT ND50 value was highest regardless of infection/vaccination status, and groups with recent antigenic stimulation showed high PRNT titers overall. Groups with double Omicron stimulation, either by BI plus BA.4/5 bivalent vaccination or repeated BI, exhibited significantly higher BA.5 and BN.1 PRNT to WT PRNT ratios than those with single Omicron stimulation. Overall group immunity was estimated to be boosted in January 2023, reflecting the effect of the BA.4/5 bivalent booster and additional BIs, but slightly declined in June 2023. A substantial increase in the antibody concentrations of IVIG products was noticed in 2022, and recently produced IVIG products exhibited a substantial level of cross-reactive neutralizing activity against emerging variants. Neutralizing activity against emerging variants could be enhanced by repeated antigenic stimulation via BI and/or updated vaccination. Overall group immunity was elevated accordingly, and IVIG products showed substantial activity against circulating strains.

RIGER analyses of RNAi arrayed screen

With the continuous emergence of highly transmissible SARS-CoV-2 variants, the comparison of their infectivity has become a critical issue for public health. However, a direct assessment of the viral characteristic has been challenging because of the lack of appropriate experimental models and efficient methods. Here, we integrated human alveolar organoids and single-cell transcriptome sequencing to facilitate the evaluation. In a proof-of-concept study with four highly transmissible SARS-CoV-2 variants, including GR (B.1.1.119), Alpha (B.1.1.7), Delta (B.1.617.2), and Omicron (BA.1), a rapid evaluation of the relative infectivity was possible. Our system demonstrates that the Omicron variant is 5- to 7-fold more infectious to human alveolar cells than the other SARS-CoV-2 variants at the initial stage of infection. To our knowledge, for the first time, this study measures the relative infectivity of the Omicron variant under multiple virus co-infection and provides new experimental procedures that can be applied to monitor emerging viral variants.

ABSTRACT Wilms tumor (WT) is the most common renal malignancy of childhood. Despite improvements in the overall survival, relapse occurs in ~15% of patients with favorable histology WT (FHWT). Half of these patients will succumb to their disease. Identifying novel targeted therapies in a systematic manner remains challenging in part due to the lack of faithful preclinical in vitro models. We established ten short-term patient-derived WT cell lines and characterized these models using low-coverage whole genome sequencing, whole exome sequencing and RNA-sequencing, which demonstrated that these ex-vivo models faithfully recapitulate WT biology. We then performed targeted RNAi and CRISPR-Cas9 loss-of-function screens and identified the nuclear export genes ( XPO1 and KPNB1 ) as strong vulnerabilities. We observed that these models are sensitive to nuclear export inhibition using the FDA approved therapeutic agent, selinexor (KPT-330). Selinexor treatment of FHWT suppressed TRIP1 3 expression, which was required for survival. We further identified in vitro and in vivo synergy between selinexor and doxorubicin, a chemotherapy used in high risk FHWT. Taken together, we identified XPO1 inhibition with selinexor as a potential therapeutic option to treat FHWTs and in combination with doxorubicin, leads to durable remissions in vivo .

Abstract A prospective cohort study was conducted for adults with a diagnosis of with coronavirus disease 2019 (COVID-19). Convalescent blood samples were obtained 4, 6, and 11 months after severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The seropositivity of anti-spike antibody was maintained in all patients (100%) until 11 months after COVID-19 diagnosis. Neutralizing antibody levels against wild-type SARS-CoV-2 gradually decreased but remained positive in >50% of patients 11 months after diagnosis: in 98.5% (67 of 68) at 4 months, 86.8% (46 of 53) at 6 months, and 58.8% (40 of 68) at 11 months. However, cross-neutralizing activity against the Beta and Delta variants was attenuated 2.53-fold and 2.93-fold, respectively, compared with the wild-type strain.