A molnupiravir-associated mutational signature in global SARS-CoV-2 genomes.

Molnupiravir, an antiviral medication widely used against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), acts by inducing mutations in the virus genome during replication. Most random mutations are likely to be deleterious to the virus and many will be lethal; thus, molnupiravir-induced elevated mutation rates reduce viral load1,2. However, if some patients treated with molnupiravir do not fully clear the SARS-CoV-2 infections, there could be the potential for onward transmission of molnupiravir-mutated viruses. Here we show that SARS-CoV-2 sequencing databases contain extensive evidence of molnupiravir mutagenesis. Using a systematic approach, we find that a specific class of long phylogenetic branches, distinguished by a high proportion of G-to-A and C-to-T mutations, are found almost exclusively in sequences from 2022, after the introduction of molnupiravir treatment, and in countries and age groups with widespread use of the drug. We identify a mutational spectrum, with preferred nucleotide contexts, from viruses in patients known to have been treated with molnupiravir and show that its signature matches that seen in these long branches, in some cases with onward transmission of molnupiravir-derived lineages. Finally, we analyse treatment records to confirm a direct association between these high G-to-A branches and the use of molnupiravir.

RETaliation-Tackling Rare Resistance Alterations to Osimertinib.

RET fusions occur as a rare mechanism of acquired resistance to osimertinib in patients with EGFR mutation-positive non-small cell lung cancer. Inhibiting RET alongside osimertinib shows promising clinical activity, but innovative approaches are needed to seek regulatory approvals in these rare treatment resistance settings. See related article by Rotow et al., p. 2979.

Olaparib for previously treated BRCA mutation-positive hormone-relapsed metastatic prostate cancer

This evaluation uses new cost-effectiveness estimates to update olaparib for previously treated BRCA mutation-positive hormone-relapsed metastatic prostate cancer (NICE technology appraisal guidance TA831). No new clinical evidence was reviewed. Treatments for BRCA mutation-positive hormone-relapsed metastatic prostate cancer that has progressed after enzalutamide or abiraterone include taxanes (for example, docetaxel or cabazitaxel), radium­223 dichloride and best supportive care. The company provided evidence based on whether or not people had already had a taxane. This is because people have different treatments depending on whether they have had a taxane before. Clinical trial evidence shows that people taking olaparib have more time before their cancer gets worse, and live longer overall, than people having retreatment with abiraterone or enzalutamide. However, this retreatment is not considered effective and is not standard care in the NHS. For people who have had a taxane before, olaparib has not been directly compared with docetaxel, cabazitaxel or radium­223 dichloride. An indirect comparison suggests that olaparib increases how long people live compared with cabazitaxel. For people who have not had a taxane before there is also no direct evidence comparing olaparib with docetaxel or best supportive care. But an exploratory indirect comparison suggests that olaparib may increase how long people live compared with both best supportive care and docetaxel. Olaparib likely meets NICE's criteria for a life-extending treatment at the end of life. When taking this into account, the most likely cost-effectiveness estimates are within what NICE considers an acceptable use of NHS resources. So olaparib is recommended.

Osimertinib and Selpercatinib Efficacy, Safety, and Resistance in a Multicenter, Prospectively Treated Cohort of EGFR-Mutant and RET Fusion-Positive Lung Cancers.

PURPOSE: Acquired RET fusions have been reported at resistance to treatment with EGFR inhibitors in EGFR-mutant non-small cell lung cancer (NSCLC); however, a multicenter cohort of patients with EGFR-mutant lung cancers treated with osimertinib and selpercatinib for RET fusion-mediated osimertinib resistance has not previously been published. PATIENTS AND METHODS: Patients who received selpercatinib in combination with osimertinib on a prospective expanded access clinical trial (NCT03906331) and single-patient compassionate use programs across five countries were centrally analyzed. All patients had advanced EGFR-mutant NSCLC with a RET fusion detected from tissue or plasma following osimertinib therapy. Clinicopathologic and outcomes data were collected. RESULTS: Fourteen patients with EGFR-mutant and RET fusion-positive lung cancers who experienced prior progression on osimertinib received osimertinib and selpercatinib. EGFR exon 19 deletions (±T790M, 86%) and non-KIF5B fusions (CCDC6-RET 50%, NCOA4-RET 36%) predominated. Osimertinib 80 mg daily and selpercatinib 80 mg twice daily were the most commonly administered dosages. The response rate, disease control rate, and median treatment duration were 50% [95% confidence interval (CI), 25%-75%, n = 12], 83% (95% CI, 55%-95%), and 7.9 months (range, 0.8-25+), respectively. Resistance was complex, involving EGFR on-target (EGFR C797S), RET on-target (RET G810S), and off-target (EML4-ALK/STRN-ALK, KRAS G12S, BRAF V600E) mechanisms; RET fusion loss; or polyclonal mechanisms. CONCLUSIONS: For patients with EGFR-mutant NSCLC with an acquired RET fusion as a mechanism of EGFR inhibitor resistance, the addition of selpercatinib to osimertinib was feasible and safe and offered clinical benefit, supporting the prospective evaluation of this combination. See related commentary by Krebs and Popat, p. 2951.

Mobocertinib for treating EGFR exon 20 insertion mutation-positive advanced non-small-cell lung cancer after platinum-based chemotherapy

Evidence-based recommendations on mobocertinib (EXKIVITY) for treating EGFR exon 20 insertion mutation-positive advanced non-small-cell lung cancer after platinum-based chemotherapy in adults. Commercial arrangement There is a simple discount patient access scheme for mobocertinib. NHS organisations can get details on the Commercial Access and Pricing (CAP) Portal. Non-NHS organisations can contact smb.marketaccessUK@takeda.com for details.

A Detouring Experience Not Recommended: Lessons Learned from PF00299804.

Patients with mutant EGFR positive non-small cell lung cancer (NSCLC) benefit from tyrosine kinase inhibitor (TKI) treatment. However, all patients ultimately develop acquired resistance, half of which are attributed to the EGFR exon 20 T790M mutation. A landmark publication in Cancer Research in 2007 demonstrated improved drug potency and pan-human EGFR (HER) inhibition with PF00299804, a second-generation EGFR TKI. Compared with first-generation EGFR TKI, PF00299804 showed the ability to overcome T790M mutation in vitro and had the potential to improve treatment outcomes of patients with mutant EGFR-positive NSCLC. Here we review the preclinical and clinical development of PF00299804 and reflect on the lessons learned from this detouring experience. See related article by Engelman and colleagues, Cancer Res 2007;67:11924-32.

Hydrogen Sulfide-Linked Persulfidation Maintains Protein Stability of ABSCISIC ACID-INSENSITIVE 4 and Delays Seed Germination.

Hydrogen sulfide (H2S) is an endogenous gaseous molecule that plays an important role in the plant life cycle. The multiple transcription factor ABSCISIC ACID INSENSITIVE 4 (ABI4) was precisely regulated to participate in the abscisic acid (ABA) mediated signaling cascade. However, the molecular mechanisms of how H2S regulates ABI4 protein level to control seed germination and seedling growth have remained elusive. In this study, we demonstrated that ABI4 controls the expression of L-CYSTEINE DESULFHYDRASE1 (DES1), a critical endogenous H2S-producing enzyme, and both ABI4 and DES1-produced H2S have inhibitory effects on seed germination. Furthermore, the ABI4 level decreased during seed germination while H2S triggered the enhancement of the persulfidation level of ABI4 and alleviated its degradation rate, which in turn inhibited seed germination and seedling establishment. Conversely, the mutation of ABI4 at Cys250 decreased ABI4 protein stability and facilitated seed germination. Moreover, ABI4 degradation is also regulated via the 26S proteasome pathway. Taken together, these findings suggest a molecular link between DES1 and ABI4 through the post-translational modifications of persulfidation during early seedling development.

Classical RAS proteins are not essential for paradoxical ERK activation induced by RAF inhibitors.

RAF inhibitors unexpectedly induce ERK signaling in normal and tumor cells with elevated RAS activity. Paradoxical activation is believed to be RAS dependent. In this study, we showed that LY3009120, a pan-RAF inhibitor, can unexpectedly cause paradoxical ERK activation in KRASG12C-dependent lung cancer cell lines, when KRAS is inhibited by ARS1620, a KRASG12C inhibitor. Using H/N/KRAS-less mouse embryonic fibroblasts, we discovered that classical RAS proteins are not essential for RAF inhibitor-induced paradoxical ERK signaling. In their absence, RAF inhibitors can induce ERK phosphorylation, ERK target gene transcription, and cell proliferation. We further showed that the MRAS/SHOC2 complex is required for this process. This study highlights the complexity of the allosteric RAF regulation by RAF inhibitors, and the importance of other RAS-related proteins in this process.

Analysis of the Knockdown Resistance Locus (kdr) in Anopheles stephensi, An. arabiensis, and Culex pipiens s.l. for Insight Into the Evolution of Target-site Pyrethroid Resistance in Eastern Ethiopia.

The malaria vector, Anopheles stephensi, which is typically restricted to South Asia and the Middle East, was recently detected in the Horn of Africa. Addressing the spread of this vector could involve integrated vector control that considers the status of insecticide resistance of multiple vector species in the region. Previous reports indicate that the knockdown resistance mutations (kdr) in the voltage-gated sodium channel (vgsc) are absent in both pyrethroid-resistant and pyrethroid-sensitive An. stephensi in eastern Ethiopia; however, similar information about other vector species in the same areas is limited. In this study, kdr and the neighboring intron were analyzed in An. stephensi, An. arabiensis, and Culex pipiens s.l. collected between 2016 and 2017 to determine the evolutionary history of kdr in eastern Ethiopia. A sequence analysis revealed that all of Cx. pipiens s.l. (N = 42) and 71.6% of the An. arabiensis (N = 67) carried kdr L1014F, which is known to confer target-site pyrethroid resistance. Intronic variation was only observed in An. stephensi (six segregating sites, three haplotypes), which was previously shown to have no kdr mutations. In addition, no evidence of non-neutral evolutionary processes was detected at the An. stephensi kdr intron, thereby further supporting the target-site mechanism not being a major resistance mechanism in this An. stephensi population. Overall, these results show key differences in the evolution of target-site pyrethroid/dichlorodiphenyltrichloroethane resistance mutations in populations of vector species from the same region. Variations in insecticide resistance mechanism profiles between eastern Ethiopian mosquito vectors may lead to different responses to insecticides used in integrated vector control.

Genetic alterations of the SUMO isopeptidase SENP6 drive lymphomagenesis and genetic instability in diffuse large B-cell lymphoma.

SUMOylation is a post-translational modification of proteins that regulates these proteins' localization, turnover or function. Aberrant SUMOylation is frequently found in cancers but its origin remains elusive. Using a genome-wide transposon mutagenesis screen in a MYC-driven B-cell lymphoma model, we here identify the SUMO isopeptidase (or deconjugase) SENP6 as a tumor suppressor that links unrestricted SUMOylation to tumor development and progression. Notably, SENP6 is recurrently deleted in human lymphomas and SENP6 deficiency results in unrestricted SUMOylation. Mechanistically, SENP6 loss triggers release of DNA repair- and genome maintenance-associated protein complexes from chromatin thereby impairing DNA repair in response to DNA damages and ultimately promoting genomic instability. In line with this hypothesis, SENP6 deficiency drives synthetic lethality to Poly-ADP-Ribose-Polymerase (PARP) inhibition. Together, our results link SENP6 loss to defective genome maintenance and reveal the potential therapeutic application of PARP inhibitors in B-cell lymphoma.

Detection of Gag C-terminal mutations among HIV-1 non-B subtypes in a subset of Cameroonian patients.

Response to ritonavir-boosted-protease inhibitors (PI/r)-based regimen is associated with some Gag mutations among HIV-1 B-clade. There is limited data on Gag mutations and their covariation with mutations in protease among HIV-1 non-B-clades at PI/r-based treatment failure. Thus, we characterized Gag mutations present in isolates from HIV-1 infected individuals treated with a PI/r-regimen (n = 143) and compared them with those obtained from individuals not treated with PI/r (ART-naïve [n = 101] or reverse transcriptase inhibitors (RTI) treated [n = 118]). The most frequent HIV-1 subtypes were CRF02_AG (54.69%), A (13.53%), D (6.35%) and G (4.69%). Eighteen Gag mutations showed a significantly higher prevalence in PI/r-treated isolates compared to ART-naïve (p < 0.05): Group 1 (prevalence < 1% in drug-naïve): L449F, D480N, L483Q, Y484P, T487V; group 2 (prevalence 1-5% in drug-naïve): S462L, I479G, I479K, D480E; group 3 (prevalence ≥ 5% in drug-naïve): P453L, E460A, R464G, S465F, V467E, Q474P, I479R, E482G, T487A. Five Gag mutations (L449F, P453L, D480E, S465F, Y484P) positively correlated (Phi ≥ 0.2, p < 0.05) with protease-resistance mutations. At PI/r-failure, no significant difference was observed between patients with and without these associated Gag mutations in term of viremia or CD4 count. This analysis suggests that some Gag mutations show an increased frequency in patients failing PIs among HIV-1 non-B clades.

Efficacy and safety of enasidenib and azacitidine combination in patients with IDH2 mutated acute myeloid leukemia and not eligible for intensive chemotherapy.

Preclinically, enasidenib and azacitidine (ENA + AZA) synergistically enhance cell differentiation, and venetoclax (VEN), a small molecule Bcl2 inhibitor (i) is particularly effective in IDH2 mutated acute myeloid leukemia (IDH2mutAML). This open label phase II trial enrolled patients (pts) with documented IDH2mutAML. All patients received AZA 75 mg/m2/d x 7 d/cycle and ENA 100 mg QD continuously. Concomitant Bcl2i and FLT3i were allowed (NCT03683433).Twenty-six pts received ENA + AZA (median 68 years, range, 24-88); 7 newly diagnosed (ND) and 19 relapsed/refractory (R/R). In R/R AML patients, three had received prior ENA and none had received prior VEN. The composite complete remission rate (CRc) [complete remission (CR) or complete remission with incomplete hematologic recovery (CRi)] was 100% in ND AML, and 58% in R/R AML. Median OS was not reached in ND AML with median follow-up of 13.1 months (mo); Pts treated in first relapse had improved OS than those with ≥2 relapse (median OS not reached vs 5.2 mo; HR 0.24, 95% CI 0.07-0.79, p = 0.04). Two patients received ENA + AZA with a concomitant FLT3i, one responding ND AML patient and one nonresponding R/R AML patient. Seven R/R AML pts received ENA + AZA + VEN triplet, and with median follow up of 11.2 mo, median OS was not reached and 6-mo OS was 70%. The most frequent treatment-emergent adverse events include febrile neutropenia (23%). Adverse events of special interest included all-grade IDH differentiation syndrome (8%) and indirect hyperbilirubinemia (35%). ENA + AZA was a well-tolerated, and effective therapy for elderly pts with IDH2mut ND AML as well as pts with R/R AML. The addition of VEN to ENA + AZA appears to improve outcomes in R/R IDH2mutAML.Clinical trial registration information: https://clinicaltrials.gov/.NCT03683433.

Effective Menin inhibitor-based combinations against AML with MLL rearrangement or NPM1 mutation (NPM1c).

Treatment with Menin inhibitor (MI) disrupts the interaction between Menin and MLL1 or MLL1-fusion protein (FP), inhibits HOXA9/MEIS1, induces differentiation and loss of survival of AML harboring MLL1 re-arrangement (r) and FP, or expressing mutant (mt)-NPM1. Following MI treatment, although clinical responses are common, the majority of patients with AML with MLL1-r or mt-NPM1 succumb to their disease. Pre-clinical studies presented here demonstrate that genetic knockout or degradation of Menin or treatment with the MI SNDX-50469 reduces MLL1/MLL1-FP targets, associated with MI-induced differentiation and loss of viability. MI treatment also attenuates BCL2 and CDK6 levels. Co-treatment with SNDX-50469 and BCL2 inhibitor (venetoclax), or CDK6 inhibitor (abemaciclib) induces synergistic lethality in cell lines and patient-derived AML cells harboring MLL1-r or mtNPM1. Combined therapy with SNDX-5613 and venetoclax exerts superior in vivo efficacy in a cell line or PD AML cell xenografts harboring MLL1-r or mt-NPM1. Synergy with the MI-based combinations is preserved against MLL1-r AML cells expressing FLT3 mutation, also CRISPR-edited to introduce mtTP53. These findings highlight the promise of clinically testing these MI-based combinations against AML harboring MLL1-r or mtNPM1.

Antibody-Drug Conjugates: A New Addition to the Treatment Landscape of EGFR-Mutant Non-Small Cell Lung Cancer.

The emergence of treatment resistance to targeted agents is currently inevitable and inherently heterogeneous in cancer, presenting significant challenges for improving survival outcomes in patients. This is not an exception for cancers harboring EGFR mutations, one of the most prevalently observed oncogenic alterations in non-small cell lung cancer (NSCLC) targeted clinically. Currently, numerous efforts have attempted to delay or overcome acquired resistance to EGFR-tyrosine kinase inhibitors (TKI), changing the treatment landscape of EGFR-mutant NSCLC. Haikala and colleagues have developed a unique strategy using patritumab deruxtecan, an antibody-drug conjugate targeting human epidermal growth factor receptor 3 (HER3) linked to exatecan derivatives, for treating EGFR-mutant NSCLC. By incorporating EGFR TKIs to upregulate surface HER3 expression, the antitumor efficacy of patritumab deruxtecan was augmented in various preclinical models. In parallel, Jänne and colleagues reported the clinical activity of patrimumab deruxtecan in patients with EGFR-mutant NSCLC with prior EGFR TKI treatment. These two studies provide the grounds for hopeful anticipation for a novel strategy that concurrently targets compensatory feedback loops in addition to oncogenic signaling pathways.See related article by Haikala et al., p. 130.

Heterogeneity of glutamine metabolism in acquired-EGFR-TKI-resistant lung cancer.

AIMS: The purpose of this study was to evaluate the heterogeneities of glutamine metabolism in EGFR-TKI-resistant lung cancer cells and its potential as a therapeutic target. MAIN METHODS: Cell proliferation and cell cycle assays was performed by IncuCyte real-time analysis and flow cytometry, respectively. Tumor growth was assessed in xenografts implanted with HCC827 GR. An isotopologue analysis was conducted by LC-MS/MS using 13C-(U)-glutamine labeling to determine the amounts of metabolites. Cellular ATP and mitochondrial oxidative phosphorylation were determined by XFp analysis. KEY FINDINGS: We found that the cell growth of the two acquired EGFR-TKI-resistant lung cancer cells lines (HCC827 GR and H292 ER) depends on glutamine. In HCC827 GR, glutamine deficiency caused reduced GSH synthesis and, subsequently, enhanced ROS generation relative to their parental cells, HCC827. On the other hand, in H292 ER, glutamine mainly acted as a carbon source for TCA-cycle intermediates, and its depletion led to reduced mitochondrial ATP production. CB-839, a specific GLS inhibitor, inhibited the latter's conversion of glutamine to glutamate and exerted enhanced anti-proliferating effects on the two acquired EGFR-TKI-resistant lung cancer cell lines versus their parental cell lines. Moreover, oral administration of CB-839 significantly suppressed HCC827 GR tumor growth in the xenograft model. SIGNIFICANCE: These findings suggest that glutamine dependency in acquired EGFR-TKI-resistant lung cancer is heterogeneous and that inhibition of glutamine metabolism by CB-839 may serve as a therapeutic tool for acquired EGFR-TKI-resistant lung cancer.

Chemical Evolution of Rhinovirus Identifies Capsid-Destabilizing Mutations Driving Low-pH-Independent Genome Uncoating.

Rhinoviruses (RVs) cause recurrent infections of the nasal and pulmonary tracts, life-threatening conditions in chronic respiratory illness patients, predisposition of children to asthmatic exacerbation, and large economic cost. RVs are difficult to treat. They rapidly evolve resistance and are genetically diverse. Here, we provide insight into RV drug resistance mechanisms against chemical compounds neutralizing low pH in endolysosomes. Serial passaging of RV-A16 in the presence of the vacuolar proton ATPase inhibitor bafilomycin A1 (BafA1) or the endolysosomotropic agent ammonium chloride (NH4Cl) promoted the emergence of resistant virus populations. We found two reproducible point mutations in viral proteins 1 and 3 (VP1 and VP3), A2526G (serine 66 to asparagine [S66N]), and G2274U (cysteine 220 to phenylalanine [C220F]), respectively. Both mutations conferred cross-resistance to BafA1, NH4Cl, and the protonophore niclosamide, as identified by massive parallel sequencing and reverse genetics, but not the double mutation, which we could not rescue. Both VP1-S66 and VP3-C220 locate at the interprotomeric face, and their mutations increase the sensitivity of virions to low pH, elevated temperature, and soluble intercellular adhesion molecule 1 receptor. These results indicate that the ability of RV to uncoat at low endosomal pH confers virion resistance to extracellular stress. The data endorse endosomal acidification inhibitors as a viable strategy against RVs, especially if inhibitors are directly applied to the airways. IMPORTANCE Rhinoviruses (RVs) are the predominant agents causing the common cold. Anti-RV drugs and vaccines are not available, largely due to rapid evolutionary adaptation of RVs giving rise to resistant mutants and an immense diversity of antigens in more than 160 different RV types. In this study, we obtained insight into the cell biology of RVs by harnessing the ability of RVs to evolve resistance against host-targeting small chemical compounds neutralizing endosomal pH, an important cue for uncoating of normal RVs. We show that RVs grown in cells treated with inhibitors of endolysosomal acidification evolved capsid mutations yielding reduced virion stability against elevated temperature, low pH, and incubation with recombinant soluble receptor fragments. This fitness cost makes it unlikely that RV mutants adapted to neutral pH become prevalent in nature. The data support the concept of host-directed drug development against respiratory viruses in general, notably at low risk of gain-of-function mutations.

Effective therapy for AML with RUNX1 mutation by cotreatment with inhibitors of protein translation and BCL2.

The majority of RUNX1 mutations in acute myeloid leukemia (AML) are missense or deletion-truncation and behave as loss-of-function mutations. Following standard therapy, AML patients expressing mtRUNX1 exhibit inferior clinical outcome than those without mutant RUNX1. Studies presented here demonstrate that as compared with AML cells lacking mtRUNX1, their isogenic counterparts harboring mtRUNX1 display impaired ribosomal biogenesis and differentiation, as well as exhibit reduced levels of wild-type RUNX1, PU.1, and c-Myc. Compared with AML cells with only wild-type RUNX1, AML cells expressing mtRUNX1 were also more sensitive to the protein translation inhibitor homoharringtonine (omacetaxine) and BCL2 inhibitor venetoclax. Homoharringtonine treatment repressed enhancers and their BRD4 occupancy and was associated with reduced levels of c-Myc, c-Myb, MCL1, and Bcl-xL. Consistent with this, cotreatment with omacetaxine and venetoclax or BET inhibitor induced synergistic in vitro lethality in AML expressing mtRUNX1. Compared with each agent alone, cotreatment with omacetaxine and venetoclax or BET inhibitor also displayed improved in vivo anti-AML efficacy, associated with improved survival of immune-depleted mice engrafted with AML cells harboring mtRUNX1. These findings highlight superior efficacy of omacetaxine-based combination therapies for AML harboring mtRUNX1.