Cooperative genomic lesions in HRAS-mutant cancers predict resistance to farnesyltransferase inhibitors.

In the clinical development of farnesyltransferase inhibitors (FTIs) for HRAS-mutant tumors, responses varied by cancer type. Co-occurring mutations may affect responses. We aimed to uncover cooperative genetic events specific to HRAS-mutant tumors and to study their effect on sensitivity to FTIs. Using targeted sequencing data from the MSK-IMPACT and Dana-Farber Cancer Institute Genomic Evidence Neoplasia Information Exchange databases, we identified comutations that were observed predominantly in HRAS-mutant versus KRAS-mutant or NRAS-mutant cancers. HRAS-mutant cancers had a higher frequency of coaltered mutations (48.8%) in the MAPK, PI3K, or RTK pathway genes, compared with KRAS-mutant (41.4%) and NRAS-mutant (38.4%) cancers (p < 0.05). Class 3 BRAF, NF1, PTEN, and PIK3CA mutations were more prevalent in HRAS-mutant lineages. To study the effects of comutations on sensitivity to FTIs, HrasG13R was transfected into "RASless" (Kraslox/lox/Hras-/-/Nras-/-/RERTert/ert) mouse embryonic fibroblasts (MEFs), which sensitized nontransfected MEFs to tipifarnib. Comutation in the form of Pten or Nf1 deletion and Pik3caH1047R transduction led to resistance to tipifarnib in HrasG13R-transfected MEFs in the presence or absence of KrasWT, whereas BrafG466E transduction led to resistance to tipifarnib only in the presence of KrasWT. Combined treatment with tipifarnib and MEK inhibition sensitized cells to tipifarnib in all settings, including in MEFs with PI3K pathway comutations. HRAS-mutant tumors demonstrate lineage-dependent MAPK or PI3K pathway alterations, which confer resistance to tipifarnib. The combined use of FTIs and MEK inhibition is a promising strategy for HRAS-mutant tumors.

RBM10 loss induces aberrant splicing of cytoskeletal and extracellular matrix mRNAs and promotes metastatic fitness.

RBM10 modulates transcriptome-wide cassette exon splicing. Loss-of-function RBM10 mutations are enriched in thyroid cancers with distant metastases. Analysis of transcriptomes and genes mis-spliced by RBM10 loss showed pro-migratory and RHO/RAC signaling signatures. RBM10 loss increases cell velocity. Cytoskeletal and ECM transcripts subject to exon-inclusion events included vinculin (VCL), tenascin C (TNC) and CD44. Knockdown of the VCL exon inclusion transcript in RBM10-null cells reduced cell velocity, whereas knockdown of TNC and CD44 exon-inclusion isoforms reduced invasiveness. RAC1-GTP levels were increased in RBM10-null cells. Mouse Hras G12V /Rbm1O KO thyrocytes develop metastases that are reversed by RBM10 or by combined knockdown of VCL, CD44 and TNC inclusion isoforms. Thus, RBM10 loss generates exon inclusions in transcripts regulating ECM-cytoskeletal interactions, leading to RAC1 activation and metastatic competency. Moreover, a CRISPR-Cas9 screen for synthetic lethality with RBM10 loss identified NFkB effectors as central to viability, providing a therapeutic target for these lethal thyroid cancers.

Quantitating SARS-CoV-2 Neutralizing Antibodies from Human Dried Blood Spots.

Background: In the earliest days of COVID-19 pandemic, the collection of dried blood spots (DBS) enabled public health laboratories to undertake population-scale seroprevalence studies to estimate rates of SARS-CoV-2 exposure. With SARS-CoV-2 seropositivity levels now estimated to exceed 94% in the United States, attention has turned to using DBS to assess functional (neutralizing) antibodies within cohorts of interest. Methods: Contrived DBS eluates from convalescent, fully vaccinated and pre-COVID-19 serum samples were evaluated in SARS-CoV-2 plaque reduction neutralization titer (PRNT) assays, a SARS-CoV-2 specific 8-plex microsphere immunoassay, a cell-based pseudovirus assay, and two different spike-ACE2 inhibition assays, an in-house Luminex-based RBD-ACE2 inhibition assay and a commercial real-time PCR-based inhibition assay (NAB-Sure™). Results: DBS eluates from convalescent individuals were compatible with the spike-ACE2 inhibition assays, but not cell-based pseudovirus assays or PRNT. However, the insensitivity of cell-based pseudovirus assays was overcome with DBS eluates from vaccinated individuals with high SARS-CoV-2 antibody titers. Conclusion: SARS-CoV-2 neutralizing titers can be derived with confidence from DBS eluates, thereby opening the door to the use of these biospecimens for the analysis of vulnerable populations and normally hard to reach communities.

Genetically encoding multiple functionalities into extracellular vesicles for the targeted delivery of biologics to T cells.

The genetic modification of T cells has advanced cellular immunotherapies, yet the delivery of biologics specifically to T cells remains challenging. Here we report a suite of methods for the genetic engineering of cells to produce extracellular vesicles (EVs)-which naturally encapsulate and transfer proteins and nucleic acids between cells-for the targeted delivery of biologics to T cells without the need for chemical modifications. Specifically, the engineered cells secreted EVs that actively loaded protein cargo via a protein tag and that displayed high-affinity T-cell-targeting domains and fusogenic glycoproteins. We validated the methods by engineering EVs that delivered Cas9-single-guide-RNA complexes to ablate the gene encoding the C-X-C chemokine co-receptor type 4 in primary human CD4+ T cells. The strategy is amenable to the targeted delivery of biologics to other cell types.

Effects of aluminum-salt, CpG and emulsion adjuvants on the stability and immunogenicity of a virus-like particle displaying the SARS-CoV-2 receptor-binding domain (RBD).

Second-generation COVID-19 vaccines with improved immunogenicity (e.g., breadth, duration) and availability (e.g., lower costs, refrigerator stable) are needed to enhance global coverage. In this work, we formulated a clinical-stage SARS-CoV-2 receptor-binding domain (RBD) virus-like particle (VLP) vaccine candidate (IVX-411) with widely available adjuvants. Specifically, we assessed the in vitro storage stability and in vivo mouse immunogenicity of IVX-411 formulated with aluminum-salt adjuvants (Alhydrogel™, AH and Adjuphos™, AP), without or with the TLR-9 agonist CpG-1018™ (CpG), and compared these profiles to IVX-411 adjuvanted with an oil-in-water nano-emulsion (AddaVax™, AV). Although IVX-411 bound both AH and AP, lower binding strength of antigen to AP was observed by Langmuir binding isotherms. Interestingly, AH- and AP-adsorbed IVX-411 had similar storage stability profiles as measured by antigen-binding assays (competitive ELISAs), but the latter displayed higher pseudovirus neutralizing titers (pNT) in mice, at levels comparable to titers elicited by AV-adjuvanted IVX-411. CpG addition to alum (AP or AH) resulted in a marginal trend of improved pNTs in stressed samples only, yet did not impact the storage stability profiles of IVX-411. In contrast, previous work with AH-formulations of a monomeric RBD antigen showed greatly improved immunogenicity and decreased stability upon CpG addition to alum. At elevated temperatures (25, 37°C), IVX-411 formulated with AH or AP displayed decreased in vitro stability compared to AV-formulated IVX-411and this rank-ordering correlated with in vivo performance (mouse pNT values). This case study highlights the importance of characterizing antigen-adjuvant interactions to develop low cost, aluminum-salt adjuvanted recombinant subunit vaccine candidates.

A transient intermediate RNA structure underlies the regulatory function of the E. coli thiB TPP translational riboswitch.

Riboswitches are cis-regulatory RNA elements that regulate gene expression in response to ligand binding through the coordinated action of a ligand-binding aptamer domain (AD) and a downstream expression platform (EP). Previous studies of transcriptional riboswitches have uncovered diverse examples that utilize structural intermediates that compete with the AD and EP folds to mediate the switching mechanism on the timescale of transcription. Here we investigate whether similar intermediates are important for riboswitches that control translation by studying the Escherichia coli thiB thiamin pyrophosphate (TPP) riboswitch. Using cellular gene expression assays, we first confirmed that the riboswitch acts at the level of translational regulation. Deletion mutagenesis showed the importance of the AD-EP linker sequence for riboswitch function. Sequence complementarity between the linker region and the AD P1 stem suggested the possibility of an intermediate nascent RNA structure called the antisequestering stem that could mediate the thiB switching mechanism. Experimentally informed secondary structure models of the thiB folding pathway generated from chemical probing of nascent thiB structures in stalled transcription elongation complexes confirmed the presence of the antisequestering stem, and showed it may form cotranscriptionally. Additional mutational analysis showed that mutations to the antisequestering stem break or bias thiB function according to whether the antisequestering stem or P1 is favored. This work provides an important example of intermediate structures that compete with AD and EP folds to implement riboswitch mechanisms.

Cooperative Genomic Lesions in HRAS-Mutant Cancers Predict Resistance to Farnesyltransferase Inhibitors.

The clinical development of farnesyltransferase inhibitors (FTI) for HRAS-mutant tumors showed mixed responses dependent on cancer type. Co-occurring mutations may affect response. We aimed to uncover cooperative genetic events specific to HRAS-mutant tumors and study their effect on FTI sensitivity. Using targeted sequencing data from MSK-IMPACT and DFCI-GENIE databases we identified co-mutations in HRAS- vs KRAS- and NRAS-mutant cancers. HRAS-mutant cancers had a higher frequency of co-altered mutations (48.8%) in MAPK, PI3K, or RTK pathways genes compared to KRAS- and NRAS-mutant cancers (41.4% and 38.4%, respectively; p < 0.05). Class 3 BRAF, NF1, PTEN, and PIK3CA mutations were more prevalent in HRAS-mutant lineages. To study the effect of comutations on FTI sensitivity, HrasG13R was transfected into 'RASless' (Kraslox/lox;Hras-/-;Nras-/-) mouse embryonic fibroblasts (MEFs) which sensitized non-transfected MEFs to tipifarnib. Comutation in the form of Pten or Nf1 deletion or Pik3caH1047R or BrafG466E transduction led to relative resistance to tipifarnib in HrasG13R MEFs in the presence or absence of KrasWT. Combined treatment of tipifarnib with MEK inhibition sensitized cells to tipifarnib, including in MEFs with PI3K pathway comutations. HRAS-mutant tumors demonstrate lineage demonstrate lineage-dependent MAPK/PI3K pathway alterations that confer relative resistance to tipifarnib. Combined FTI and MEK inhibition is a promising combination for HRAS-mutant tumors.

Nanoalum Formulations Containing Aluminum Hydroxide and CpG 1018TM Adjuvants: The Effect on Stability and Immunogenicity of a Recombinant SARS-CoV-2 RBD Antigen.

Aluminum-salt vaccine adjuvants (alum) are commercially available as micron-sized particles with varying chemical composition and crystallinity. There are reports of enhanced adjuvanticity when the alum's particle size is reduced to the nanometer range. Previously, we demonstrated that a recombinant receptor-binding domain (RBD)-based COVID-19 vaccine candidate (RBD-J; RBD-L452K-F490W) formulated with aluminum hydroxide (Alhydrogel®; AH) and CpG 1018™ (CpG) adjuvants induced potent neutralizing antibody responses in mice yet displayed instability during storage. In this work, we evaluated whether sonication of AH to the nanometer size range (nanoAH) could further enhance immunogenicity or improve storage stability of the above formulation. The addition of CpG to nanoAH (at mouse doses), however, caused re-agglomeration of nanoAH. AH-CpG interactions were evaluated by Langmuir binding isotherms and zeta potential measurements, and stabilized nanoAH + CpG formulations of RBD-J were then designed by (1) optimizing CpG:Aluminum dose ratios or (2) adding a small-molecule polyanion (phytic acid, PA). Compared with the micron-sized AH + CpG formulation, the two stabilized nanoAH + CpG formulations of RBD-J demonstrated no enhancement in SARS-CoV-2 pseudovirus neutralizing titers in mice, but the PA-containing nanoAH + CpG formulation showed improved RBD-J storage stability trends (at 4, 25, and 37 °C). The formulation protocols presented herein can be employed to evaluate the potential benefits of the nanoAH + CpG adjuvant combination with other vaccine antigens in different animal models.

A ligand-gated strand displacement mechanism for ZTP riboswitch transcription control.

Cotranscriptional folding is an obligate step of RNA biogenesis that can guide RNA structure formation and function through transient intermediate folds. This process is particularly important for transcriptional riboswitches in which the formation of ligand-dependent structures during transcription regulates downstream gene expression. However, the intermediate structures that comprise cotranscriptional RNA folding pathways, and the mechanisms that enable transit between them, remain largely unknown. Here, we determine the series of cotranscriptional folds and rearrangements that mediate antitermination by the Clostridium beijerinckii pfl ZTP riboswitch in response to the purine biosynthetic intermediate ZMP. We uncover sequence and structural determinants that modulate an internal RNA strand displacement process and identify biases within natural ZTP riboswitch sequences that promote on-pathway folding. Our findings establish a mechanism for pfl riboswitch antitermination and suggest general strategies by which nascent RNA molecules navigate cotranscriptional folding pathways.

Effects of high-dose microbeam irradiation on tumor microvascular function and angiogenesis.

Microbeam radiation therapy (MRT) is a form of cancer treatment in which a single large dose of radiation is spatially fractionated in-line or grid-like patterns. Preclinical studies have demonstrated that MRT is capable of eliciting high levels of tumor response while sparing normal tissue that is exposed to the same radiation field. Since a large fraction of the MRT-treated tumor is in the dose valley region that is not directly irradiated, tumor response may be driven by radiation bystander effects, which in turn elicit a microvascular response. Differential alterations in hemodynamics between the tumor and normal tissue may explain the therapeutic advantages of MRT. Direct observation of these dynamic responses presents a challenge for conventional ex vivo analysis. Furthermore, knowledge gleaned from in vitro studies of radiation bystander response has not been widely incorporated into in vivo models of tumor radiotherapy, and the biological contribution of the bystander effect within the tumor microenvironment is unknown. In this study, we employed noninvasive, serial observations of the tumor microenvironment to address the question of how tumor vasculature and HIF-1 expression are affected by microbeam radiotherapy. Tumors (approximately 4 mm in diameter) grown in a dorsal window chamber were irradiated in a single fraction using either a single, microplanar beam (300 micron wide swath) or a wide-field setup (whole-window chamber) to a total dose of 50 Gy. The tumors were optically observed daily for seven days postirradiation. Microvascular changes in the tumor and surrounding normal tissue differed greatly between the wide-field and microbeam treatments. We present evidence that these changes may be due to dissimilar spatial and temporal patterns of HIF-1 expression induced through radiation bystander effects.

Genetic basis of murine antibacterial defense to streptococcal lung infection.

To evaluate the effect of genetic background on antibacterial defense to streptococcal infection, eight genetically diverse strains of mice (A/J, DBA/2J, CAST/Ei, FVB/NJ, BALB/cJ, C57BL/6J, 129/SvImJ, and C3H/HeJ) and tlr2-deficient mice (C57BL/6(tlr2-/-)) were infected with three doses of Streptococcus zooepidemicus (500, 5,000, or 50,000 colony-forming units) by alveolar challenge. There was a range of susceptibility between the strains at each dose and time point (6, 24, and 96 h). At the lowest dose, the 129/SvImJ and C3H/HeJ strains had significantly higher bacterial counts at all time points after infection, when compared to A/J, DBA/2J, CAST/Ei, FVB/NJ, which were resistant to infection at the low dose of innoculum. At the medium dose, 129/SvImJ and C3H/HeJ had higher bacterial counts, while A/J, DBA/2J, and BALB/cJ showed reduced streptococcal growth. After the highest dose of Streptococcus, there were minimal differences between strains, suggesting the protective impact of modifier genes can be overcome. TLR2-deficient animals contained increased bacterial load with reduced cytokines after 96 h when compared to C57BL/6J controls suggesting a role of innate immunity in late antibacterial defense. Overall, we identify vulnerable (129/SvlmJ and C3H/HeJ) and resistant (A/J, FVB, and DBA) mouse strains to streptococcal lung infection, which demonstrate divergent genetic expression profiles. These results demonstrate that innate differences in pulmonary host defense to S. zooepidemicus are dependent on host genetic factors.

Gene expression profiling of familial and sporadic interstitial pneumonia.

RATIONALE: Idiopathic interstitial pneumonia (IIP) and its familial variants are progressive and largely untreatable disorders with poorly understood molecular mechanisms. Both the genetics and the histologic type of IIP play a role in the etiology and pathogenesis of interstitial lung disease, but transcriptional signatures of these subtypes are unknown. OBJECTIVES: To evaluate gene expression in the lung tissue of patients with usual interstitial pneumonia or nonspecific interstitial pneumonia that was either familial or nonfamilial in origin, and to compare it with gene expression in normal lung parenchyma. METHODS: We profiled RNA from the lungs of 16 patients with sporadic IIP, 10 with familial IIP, and 9 normal control subjects on a whole human genome oligonucleotide microarray. RESULTS: Significant transcriptional differences exist in familial and sporadic IIPs. The genes distinguishing the genetic subtypes belong to the same functional categories as transcripts that distinguish IIP from normal samples. Relevant categories include chemokines and growth factors and their receptors, complement components, genes associated with cell proliferation and death, and genes in the Wnt pathway. The role of the chemokine CXCL12 in disease pathogenesis was confirmed in the murine bleomycin model of lung injury, with C57BL/6(CXCR4+/-) mice demonstrating significantly less collagen deposition than C57BL/6(CXCR4+/+) mice. Whereas substantial differences exist between familial and sporadic IIPs, we identified only minor gene expression changes between usual interstitial pneumonia and nonspecific interstitial pneumonia. CONCLUSIONS: Taken together, our findings indicate that differences in gene expression profiles between familial and sporadic IIPs may provide clues to the etiology and pathogenesis of IIP.

Genetic basis of murine responses to hyperoxia-induced lung injury.

To evaluate the effect of genetic background on oxygen (O2) toxicity, nine genetically diverse mouse strains (129/SvIm, A/J, BALB/cJ, BTBR+(T)/tf/tf, CAST/Ei, C3H/HeJ, C57BL/6J, DBA/2J, and FVB/NJ) were exposed to more than 99% O2 for 72 h. Immediately following the hyperoxic challenge, the mouse strains demonstrated distinct pathophysiologic responses. The BALB/cJ and CAST/Ei strains, which were the only strains to demonstrate mortality from the hyperoxic challenges, were also the only strains to display significant neutrophil infiltration into their lower respiratory tract. In addition, the O2-challenged BALB/cJ and CAST/Ei mice were among six strains (A/J, BALB/cJ, CAST/Ei, BTBR+(T)/tf/tf, DBA/2J, and C3H/HeJ) that had significantly increased interleukin 6 concentrations in the whole lung lavage fluid and were among all but one strain that had large increases in lung permeability compared with air-exposed controls. In contrast, the DBA/2J strain was the only strain not to have any significant alterations in lung permeability following hyperoxic challenge. The expression of the extracellular matrix proteins, including collagens I, III, and IV, fibronectin I, and tenascin C, also varied markedly among the mouse strains, as did the activities of total superoxide dismutase (SOD) and manganese-SOD (Mn-SOD or SOD2). These data suggest that the response to O2 depends, in part, on the genetic background and that some of the strains analyzed can be used to identify specific loci and genes underlying the response to O2.

The transcriptional response to lipopolysaccharide reveals a role for interferon-gamma in lung neutrophil recruitment.

Neutrophil recruitment to the lung after lipopolysaccharide (LPS; endotoxin) inhalation is primarily dependent on Toll-like receptor 4 (Tlr4) signaling, because it is virtually absent in mice deficient in Tlr4. However, among strains wild type for Tlr4, the magnitude of neutrophil recruitment to the lung after LPS inhalation is variable, suggesting the involvement of genes other than Tlr4. To identify genes associated with the inflammatory response to inhaled LPS, we evaluated the transcriptional response in lungs of 12 inbred strains of mice, 8 which are wild type for Tlr4 and 4 of which lack functional Tlr4. Using the promoter integration in microarray analysis algorithm, we scanned our gene list for transcription factor-binding sites significantly overrepresented among Tlr4 wild-type strains with high neutrophil influx in the lung after LPS inhalation. This analysis identified the interferon (IFN)-stimulated response element (ISRE) as the most overrepresented transcription factor (present in 24% of the promoters) associated with the neutrophil influx to the lower respiratory tract. To test the validity of this observation, we evaluated IFN-gamma-deficient mice and found that the presence of IFN-gamma is essential for robust neutrophil recruitment to the lower respiratory tract and modulation of key regulatory cytokines and chemokines after LPS inhalation. In conclusion, using a genomic approach, we identified the ISRE as a transcriptional element associated with the neutrophil response to inhaled LPS and demonstrated for the first time that IFN-gamma plays a critical role in LPS-induced neutrophil recruitment to the lower airways.

Multistrain genetic comparisons reveal CCR5 as a receptor involved in airway hyperresponsiveness.

Asthma is a ubiquitous disease with a broad range of clinical phenotypes. To better understand the complex genetic and environmental interactions underlying asthma, we compared the gene-gene interactions of four genetically distinct mouse strains that demonstrate biologically distinct responses to allergen. Using DNA microarrays and knock-out mouse studies, we showed that CCR5 plays a definitive role in the development of ovalbumin-induced allergic airway inflammatory disease. In addition, gene expression profiling data have revealed other potential novel targets for therapeutics-based research and has enhanced the understanding of the molecular mechanisms underlying the etiology of "asthma."

Spontaneous mutations in recombinant inbred mice: mutant toll-like receptor 4 (Tlr4) in BXD29 mice.

Recombinant inbred (RI) mice are frequently used to identify QTL that underlie differences in measurable phenotypes between two inbred strains of mice. Here we show that one RI strain, C57BL/6J x DBA/2J (BXD29), does not develop an inflammatory response following inhalation of LPS. Approximately 25% of F2 mice [F1(BXD29 x DBA/2J) x F1] are also unresponsive to inhaled LPS, suggesting the presence of a recessive mutation in the BXD29 strain. A genomic scan of these F2 mice revealed that unresponsive animals, but not responsive animals, are homozygous for C57BL/6J DNA at a single locus on chromosome 4 close to the genomic location of Tlr4. All progeny between BXD29 and gene-targeted Tlr4-deficient mice are unresponsive to inhaled LPS, suggesting that the mutation in the BXD29 strain is allelic with Tlr4. Moreover, the intact Tlr4 receptor is not displayed on the cell surface of BXD29 macrophages. Finally, a molecular analysis of the Tlr4 gene in BXD29 mice revealed that it is interrupted by a large insertion of repetitive DNA. These findings explain the unresponsiveness of BXD29 mice to LPS and suggest that data from BXD29 mice should not be included when using BXD mice to study phenotypes affected by Tlr4 function. Our results also suggest that the frequency of such unidentified, spontaneously occurring mutations is an issue that should be considered when RI strains are used to identify QTL.